Sample records for melanoma cell line

  1. 3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines.

    PubMed

    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.

  2. 3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines

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

    Qin, J.-Z.; Xin, H.; Nickoloff, B.J., E-mail: bnickol@lumc.edu

    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 cellmore » 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.« less

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

    PubMed

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

    2004-03-25

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

  4. Authentication of M14 melanoma cell line proves misidentification of MDA‐MB‐435 breast cancer cell line

    PubMed Central

    Korch, Christopher; Hall, Erin M.; Dirks, Wilhelm G.; Ewing, Margaret; Faries, Mark; Varella‐Garcia, Marileila; Robinson, Steven; Storts, Douglas; Turner, Jacqueline A.; Wang, Ying; Burnett, Edward C.; Healy, Lyn; Kniss, Douglas; Neve, Richard M.; Nims, Raymond W.; Reid, Yvonne A.; Robinson, William A.

    2017-01-01

    A variety of analytical approaches have indicated that melanoma cell line UCLA‐SO‐M14 (M14) and breast carcinoma cell line MDA‐MB‐435 originate from a common donor. This indicates that at some point in the past, one of these cell lines became misidentified, meaning that it ceased to correspond to the reported donor and instead became falsely identified (through cross‐contamination or other means) as a cell line from a different donor. Initial studies concluded that MDA‐MB‐435 was the misidentified cell line and M14 was the authentic cell line, although contradictory evidence has been published, resulting in further confusion. To address this question, we obtained early samples of the melanoma cell line (M14), a lymphoblastoid cell line from the same donor (ML14), and donor serum preserved at the originator's institution. M14 samples were cryopreserved in December 1975, before MDA‐MB‐435 cells were established in culture. Through a series of molecular characterizations, including short tandem repeat (STR) profiling and cytogenetic analysis, we demonstrated that later samples of M14 and MDA‐MB‐435 correspond to samples of M14 frozen in 1975, to the lymphoblastoid cell line ML14, and to the melanoma donor's STR profile, sex and blood type. This work demonstrates conclusively that M14 is the authentic cell line and MDA‐MB‐435 is misidentified. With clear provenance information and authentication testing of early samples, it is possible to resolve debates regarding the origins of problematic cell lines that are widely used in cancer research. PMID:28940260

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

    PubMed Central

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

    2012-01-01

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

  6. Fucoidan Does Not Exert Anti-Tumorigenic Effects on Uveal Melanoma Cell Lines

    PubMed Central

    Dithmer, Michaela; Kirsch, Anna-Maria; Richert, Elisabeth; Fuchs, Sabine; Wang, Fanlu; Schmidt, Harald; Coupland, Sarah E.; Roider, Johann; Klettner, Alexa

    2017-01-01

    Background. The polysaccharide fucoidan is widely investigated as an anti-cancer agent. Here, we tested the effect of fucoidan on uveal melanoma cell lines. Methods. The effect of 100 µM fucoidan was investigated on five cell lines (92.1, Mel270 OMM1, OMM2.3, OMM2.5) and of 1 µg/mL–1 mg/mL fucoidan in two cell lines (OMM1, OMM2.3). Cell proliferation and viability were investigated with a WST-1 assay, migration in a wound healing (scratch) assay. Vascular Endothelial Growth Factor (VEGF) was measured in ELISA. Angiogenesis was evaluated in co-cultures with endothelial cells. Cell toxicity was induced by hydrogen-peroxide. Protein expression (Akt, ERK1/2, Bcl-2, Bax) was investigated in Western blot. Results. Fucoidan increased proliferation in two and reduced it in one cell line. Migration was reduced in three cell lines. The effect of fucoidan on VEGF was cell type and concentration dependent. In endothelial co-culture with 92.1, fucoidan significantly increased tubular structures. Moreover, fucoidan significantly protected all tested uveal melanoma cell lines from hydrogen-peroxide induced cell death. Under oxidative stress, fucoidan did not alter the expression of Bcl-2, Bax or ERK1/2, while inducing Akt expression in 92.1 cells but not in any other cell line. Conclusion. Fucoidan did not show anti-tumorigenic effects but displayed protective and pro-angiogenic properties, rendering fucoidan unsuitable as a potential new drug for the treatment of uveal melanoma. PMID:28640204

  7. Uveal Melanoma Cell Lines: Where do they come from? (An American Ophthalmological Society Thesis).

    PubMed

    Jager, Martine J; Magner, J Antonio Bermudez; Ksander, Bruce R; Dubovy, Sander R

    2016-08-01

    To determine whether some of the most often used uveal melanoma cell lines resemble their original tumor. Analysis of the literature, patient charts, histopathology, mutations, chromosome status, HLA type, and expression of melanocyte markers on cell lines and their primary tumors. We examined five cell lines and the primary tumors from which they were derived. Four of the five examined primary tumors were unusual: one occupied the orbit, two were recurrences after prior irradiation, and one developed in an eye with a nevus of Ota. One cell line did not contain the GNA11 mutation, but it was present in the primary tumor. Three of the primary tumors had monosomy 3 (two of these lacked BAP1 expression); however, all five cell lines showed disomy 3 and BAP1 expression. All of the cell lines had gain of 8q. Two cell lines lacked expression of melanocyte markers, although these were present in the corresponding primary tumor. All cell lines could be traced back to their original uveal melanoma. Four of the five primary tumors were unusual. Cell lines often differed from their primary tumor in chromosome status and melanocyte markers. However, their specific chromosome aberrations and capacity to continue proliferation characterize them as uveal melanoma cell lines.

  8. Uveal Melanoma Cell Lines: Where do they come from? (An American Ophthalmological Society Thesis)

    PubMed Central

    Jager, Martine J.; Magner, J. Antonio Bermudez; Ksander, Bruce R.; Dubovy, Sander R.

    2016-01-01

    Purpose To determine whether some of the most often used uveal melanoma cell lines resemble their original tumor. Methods Analysis of the literature, patient charts, histopathology, mutations, chromosome status, HLA type, and expression of melanocyte markers on cell lines and their primary tumors. We examined five cell lines and the primary tumors from which they were derived. Results Four of the five examined primary tumors were unusual: one occupied the orbit, two were recurrences after prior irradiation, and one developed in an eye with a nevus of Ota. One cell line did not contain the GNA11 mutation, but it was present in the primary tumor. Three of the primary tumors had monosomy 3 (two of these lacked BAP1 expression); however, all five cell lines showed disomy 3 and BAP1 expression. All of the cell lines had gain of 8q. Two cell lines lacked expression of melanocyte markers, although these were present in the corresponding primary tumor. Conclusions All cell lines could be traced back to their original uveal melanoma. Four of the five primary tumors were unusual. Cell lines often differed from their primary tumor in chromosome status and melanocyte markers. However, their specific chromosome aberrations and capacity to continue proliferation characterize them as uveal melanoma cell lines. PMID:28018010

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

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

    Sustarsic, Elahu G.; Department of Biological Sciences, Ohio University, Athens, OH; Junnila, Riia K.

    2013-11-08

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

  10. Reduced GNG2 expression levels in mouse malignant melanomas and human melanoma cell lines

    PubMed Central

    Yajima, Ichiro; Kumasaka, Mayuko Y; Naito, Yuji; Yoshikawa, Toshikazu; Takahashi, Hiro; Funasaka, Yoko; Suzuki, Tamio; Kato, Masashi

    2012-01-01

    Heterotrimeric G protein is composed of a Gα-subunit and a Gβγ-dimer. Previous studies have revealed that Gβγ-dimers including the Gγ2 subunit (Gng2/GNG2) are associated with cell proliferation, differentiation, invasion and angiogenesis. At present, however, there is no information on the expression level of Gng2/GNG2 alone in any kind of tumor. In this study, we performed DNA microarray analysis in a benign melanocytic tumor and a malignant melanoma from RET-transgenic mice (RET-mice). Gng2 transcript expression levels in a malignant melanoma were less than 1/10 of the level in a benign tumor. The difference in Gng2 transcript expression levels between benign tumors and malignant melanomas was greatest among all of the G protein γ subunits examined in this study. Moreover, protein expression levels of Gng2 were decreased in malignant melanomas compared with those in benign melanocytic tumors in RET-mice. Analysis of human malignant melanomas also showed reduced GNG2 protein expression levels in five human malignant melanoma cell lines compared with the expression levels in normal human epithelial melanocytes (NHEM). Thus, we demonstrated for the first time that Gng2/GNG2 expression levels are reduced in malignant melanoma, suggesting that GNG2 could be a novel biomarker for malignant melanoma. PMID:22679562

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

    PubMed

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

    2003-03-01

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

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

    PubMed

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

    2016-08-01

    Cancer is a public health problem and the second leading cause of death worldwide. The incidence of cutaneous melanoma has been notably increasing, resulting in high aggressiveness and poor survival rates. Taking into account the antitumor activity of biflorin, a substance isolated from Capraria biflora L. roots that is cytotoxic in vitro and in vivo, this study aimed to demonstrate the action of biflorin against three established human melanoma cell lines that recapitulate the molecular landscape of the disease in terms of genetic alterations and mutations, such as the TP53, NRAS and BRAF genes. The results presented here indicate that biflorin reduces the viability of melanoma cell lines by DNA interactions. Biflorin causes single and double DNA strand breaks, consequently inhibiting cell cycle progression, replication and DNA repair and promoting apoptosis. Our data suggest that biflorin could be considered as a future therapeutic option for managing melanoma. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  15. The effect of ultraviolet radiation on choroidal melanocytes and melanoma cell lines: cell survival and matrix metalloproteinase production.

    PubMed

    Lai, Kenneth; Di Girolamo, Nick; Conway, Robert M; Jager, Martine J; Madigan, Michele C

    2007-05-01

    Ultraviolet radiation (UVR) can induce DNA damage and regulate the expression of factors important for tumour growth and metastasis, including matrix metalloproteinases (MMPs). Epidemiological studies suggest that chronic UVR exposure, especially during early adulthood, may be a risk factor in patients with choroidal melanoma. However, the effects of UV(R)-B on human choroidal melanocyte survival and growth are unknown. In this study, we investigated if UV(R)-B affected the in vitro survival, growth and MMP production of choroidal melanocytes and melanoma cells. Cultures of primary choroidal melanocytes and melanoma cell lines (OCM-1 and OCM-8) were exposed to UV(R)-B (0-30 mJ/cm(2)). The cell morphology and growth were examined, and cell viability was assessed using an MTT assay. Gelatin zymography was used to assess the enzymatic activity for MMP-2 and -9 in conditioned media following UV(R)-B treatment. UV(R)-B > or =20 mJ/cm(2) was cytotoxic for choroidal melanocytes. Cytotoxic doses of 5 to 10 mJ/cm(2) were found for OCM-8 and OCM-1 melanoma cell lines. Low levels of UV(R)-B (2.5 and 3.5 mJ/cm(2)) significantly reduced melanoma cell viability after 48 h, although melanocyte viability was not affected by doses of UV(R)-B <10 mJ/cm(2). Conditioned media from melanoma cells and melanocytes displayed pro-MMP-2 activity independent of UV(R)-B. Control and UV(R)-B-treated OCM-1 cells secreted active MMP-2 up to 72 h. Pro-MMP-9 activity was seen from 36 h for control and UV(R)-B-treated OCM-1 and OCM-8 cells. Melanocytes appeared more resistant to physiological doses of UV(R)-B than melanoma cells; the potential of melanocytes to initially survive DNA damage following UV(R)-B exposure may be relevant to the subsequent transformation of melanocytes to melanomas. Although UV(R)-B did not induce the production and/or activation of MMP-2 and -9 in melanocytes or melanoma cells, we are currently investigating whether DNA damage-response genes such as p53 and p21 can be

  16. Differential sensitivity of melanoma cell lines with BRAFV600E mutation to the specific Raf inhibitor PLX4032

    PubMed Central

    2010-01-01

    Blocking oncogenic signaling induced by the BRAFV600E mutation is a promising approach for melanoma treatment. We tested the anti-tumor effects of a specific inhibitor of Raf protein kinases, PLX4032/RG7204, in melanoma cell lines. PLX4032 decreased signaling through the MAPK pathway only in cell lines with the BRAFV600E mutation. Seven out of 10 BRAFV600E mutant cell lines displayed sensitivity based on cell viability assays and three were resistant at concentrations up to 10 μM. Among the sensitive cell lines, four were highly sensitive with IC50 values below 1 μM, and three were moderately sensitive with IC50 values between 1 and 10 μM. There was evidence of MAPK pathway inhibition and cell cycle arrest in both sensitive and resistant cell lines. Genomic analysis by sequencing, genotyping of close to 400 oncogeninc mutations by mass spectrometry, and SNP arrays demonstrated no major differences in BRAF locus amplification or in other oncogenic events between sensitive and resistant cell lines. However, metabolic tracer uptake studies demonstrated that sensitive cell lines had a more profound inhibition of FDG uptake upon exposure to PLX4032 than resistant cell lines. In conclusion, BRAFV600E mutant melanoma cell lines displayed a range of sensitivities to PLX4032 and metabolic imaging using PET probes can be used to assess sensitivity. PMID:20406486

  17. Cross-Priming of Naive Cd8 T Cells against Melanoma Antigens Using Dendritic Cells Loaded with Killed Allogeneic Melanoma Cells

    PubMed Central

    Berard, Frederic; Blanco, Patrick; Davoust, Jean; Neidhart-Berard, Eve-Marie; Nouri-Shirazi, Mahyar; Taquet, Nicolas; Rimoldi, Donata; Cerottini, Jean Charles; Banchereau, Jacques; Palucka, A. Karolina

    2000-01-01

    The goal of tumor immunotherapy is to elicit immune responses against autologous tumors. It would be highly desirable that such responses include multiple T cell clones against multiple tumor antigens. This could be obtained using the antigen presenting capacity of dendritic cells (DCs) and cross-priming. That is, one could load the DC with tumor lines of any human histocompatibility leukocyte antigen (HLA) type to elicit T cell responses against the autologous tumor. In this study, we show that human DCs derived from monocytes and loaded with killed melanoma cells prime naive CD45RA+CD27+CD8+ T cells against the four shared melanoma antigens: MAGE-3, gp100, tyrosinase, and MART-1. HLA-A201+ naive T cells primed by DCs loaded with HLA-A201− melanoma cells are able to kill several HLA-A201+ melanoma targets. Cytotoxic T lymphocyte priming towards melanoma antigens is also obtained with cells from metastatic melanoma patients. This demonstration of cross-priming against shared tumor antigens builds the basis for using allogeneic tumor cell lines to deliver tumor antigens to DCs for vaccination protocols. PMID:11104796

  18. Association of MITF and other melanosome-related proteins with chemoresistance in melanoma tumors and cell lines.

    PubMed

    Hertzman Johansson, Carolina; Azimi, Alireza; Frostvik Stolt, Marianne; Shojaee, Seyedmehdi; Wiberg, Henning; Grafström, Eva; Hansson, Johan; Egyházi Brage, Suzanne

    2013-10-01

    Previous studies in cell lines have suggested a role for melanosomes and related protein trafficking pathways in melanoma drug response. We have investigated the expression of six proteins related to melanosomes and melanogenesis (MITF, GPR143, gp100/PMEL, MLANA, TYRP1, and RAB27A) in pretreatment metastases from melanoma patients (n = 52) with different response to dacarbazine/temozolomide. Microphthalmia-associated transcription factor (MITF) and G-protein coupled receptor 143 (GPR143) showed significantly higher expression in nonresponders compared with responders. The premelanosome protein (gp100/PMEL) has been indicated previously in resistance to cisplatin in melanoma cells, but the expression levels of gp100/PMEL showed no association with response to dacarbazine/temozolomide in our clinical material. We also investigated the effects on chemosensitivity of siRNA inhibition of gp100/PMEL in the MNT-1 melanoma cell line. As expected from the study of the tumor material, no effect was detected with respect to response to temozolomide. However, knockdown of gp100/PMEL sensitized the cells to both paclitaxel and cisplatin. Overall, our results suggest that MITF, and several MITF-regulated factors, are associated with resistance to chemotherapy in melanoma and that different MITF targets can be of importance for different drugs.

  19. Interleukins 1alpha and 1beta secreted by some melanoma cell lines strongly reduce expression of MITF-M and melanocyte differentiation antigens.

    PubMed

    Kholmanskikh, Olga; van Baren, Nicolas; Brasseur, Francis; Ottaviani, Sabrina; Vanacker, Julie; Arts, Nathalie; van der Bruggen, Pierre; Coulie, Pierre; De Plaen, Etienne

    2010-10-01

    We report that melanoma cell lines expressing the interleukin-1 receptor exhibit 4- to 10-fold lower levels of mRNA of microphthalmia-associated transcription factor (MITF-M) when treated with interleukin-1beta. This effect is NF-kappaB and JNK-dependent. MITF-M regulates the expression of melanocyte differentiation genes such as MLANA, tyrosinase and gp100, which encode antigens recognized on melanoma cells by autologous cytolytic T lymphocytes. Accordingly, treating some melanoma cells with IL-1beta reduced by 40-100% their ability to activate such antimelanoma cytolytic T lymphocytes. Finally, we observed large amounts of biologically active IL-1alpha or IL-1beta secreted by two melanoma cell lines that did not express MITF-M, suggesting an autocrine MITF-M downregulation. We estimate that approximately 13% of melanoma cell lines are MITF-M-negative and secrete IL-1 cytokines. These results indicate that the repression of melanocyte-differentiation genes by IL-1 produced by stromal cells or by tumor cells themselves may represent an additional mechanism of melanoma immune escape.

  20. Assessment of biological effectiveness of boron neutron capture therapy in primary and metastatic melanoma cell lines.

    PubMed

    Rossini, Andrés E; Dagrosa, Maria A; Portu, Agustina; Saint Martin, Giselle; Thorp, Silvia; Casal, Mariana; Navarro, Aimé; Juvenal, Guillermo J; Pisarev, Mario A

    2015-01-01

    In order to optimize the effectiveness of Boron Neutron Capture Therapy (BNCT), Relative Biological Effectiveness (RBE) and Compound Biological Effectiveness (CBE) were determined in two human melanoma cell lines, M8 and Mel-J cells, using the amino acid p-boronophenylalanine (BPA) as boron carrier. The effects of BNCT on the primary amelanotic cell line M8 and on the metastatic pigmented melanoma cell line Mel-J were studied using colony formation assay. The RBE values were determined using both a gamma ray source, and the neutron beam from the Nuclear Reactor of the National Atomic Energy Commission (RA-3). For the determination of the RBE, cells were irradiated with increasing doses of both sources, between 1 and 8 Gy; and for the determination of CBE factors, the cells were pre-incubated with BPA before irradiation. Afterwards, the cell surviving fraction (SF) was determined for each treatment. Marked differences were observed between both cell lines. Mel-J cells were more radioresistant than the M8 cell line. The clonogenic assays showed that for a SF of 1%, the RBE values were 1.3 for M8 cells and 1.5 for Mel-J cells. Similarly, the CBE values for a 1% SF were 2.1 for M8 and 3 for Mel-J cell lines. For the endpoint of 0.1% of SF the RBE values obtained were 1.2 for M8 and 1.4 for Mel-J cells. Finally, CBE values calculated for a 0.1% were 2 and 2.6 for M8 and Mel-J cell lines respectively. In order to estimate the uptake of the non-radioactive isotope Boron 10 ((10)B), a neutron induced autoradiographic technique was performed showing discrepancies in (10)B uptake between both cell lines. These obtained in vitro results are the first effectiveness factors determined for human melanoma at the RA-3 nuclear reactor and show that BNCT dosimetry planning for patients could be successfully performed using these new factors.

  1. c-RET Molecule in Malignant Melanoma from Oncogenic RET-Carrying Transgenic Mice and Human Cell Lines

    PubMed Central

    Takeda, Kozue; Iida, Machiko; Kumasaka, Mayuko; Matsumoto, Yoshinari; Kato, Masashi

    2010-01-01

    Malignant melanoma is one of the most aggressive cancers and its incidence worldwide has been increasing at a greater rate than that of any other cancer. We previously reported that constitutively activated RFP-RET-carrying transgenic mice (RET-mice) spontaneously develop malignant melanoma. In this study, we showed that expression levels of intrinsic c-Ret, glial cell line-derived neurotrophic factor (Gdnf) and Gdnf receptor alpha 1 (Gfra1) transcripts in malignant melanomas from RET-transgenic mice were significantly upregulated compared with those in benign melanocytic tumors. These results suggest that not only introduced oncogenic RET but also intrinsic c-Ret/Gdnf are involved in murine melanomagenesis in RET-mice. We then showed that c-RET and GDNF transcript expression levels in human malignant melanoma cell lines (HM3KO and MNT-1) were higher than those in primary cultured normal human epithelial melanocytes (NHEM), while GFRa1 transcript expression levels were comparable among NHEM, HM3KO and MNT-1. We next showed c-RET and GFRa1 protein expression in HM3KO cells and GDNF-mediated increased levels of their phosphorylated c-RET tyrosine kinase and signal transduction molecules (ERK and AKT) sited potentially downstream of c-RET. Taken together with the finding of augmented proliferation of HM3KO cells after GDNF stimulation, our results suggest that GDNF-mediated c-RET kinase activation is associated with the pathogenesis of malignant melanoma. PMID:20422010

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

    PubMed

    Ohshima, Yuichiro; Yajima, Ichiro; Takeda, Kozue; Iida, Machiko; Kumasaka, Mayuko; Matsumoto, Yoshinari; Kato, Masashi

    2010-04-21

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

  3. Standardized Cannabis sativa extract attenuates tau and stathmin gene expression in the melanoma cell line.

    PubMed

    Vaseghi, Golnaz; Taki, Mohamad Javad; Javanmard, Shaghayegh Haghjooy

    2017-10-01

    Metastasis is the main cause of death in patients with melanoma. Cannabis-based medicines are effective adjunctive drugs in cancer patients. Tau and Stathmin proteins are the key proteins in cancer metastasis. Here we have investigated the effect of a standardized Cannabis sativa extract on cell migration and Tau and Stathmin gene expression in the melanoma cell line. In the treatment group, melanoma (B1617) was treated 48 hr with various concentrations of standardized C. sativa extract. Cells with no treatment were considered as the control group, then study was followed by Quantitative RT-Real Time PCR assay. Relative gene expression was calculated by the ΔΔct method. Migration assay was used to evaluate cancer metastasis. Tau and stathmin gene expression was significantly decreased compared to the control group. Cell migration was also significantly reduced compared to controls. C. sativa decreased tau and stathmin gene expression and cancer metastasis. The results may have some clinical relevance for the use of cannabis-based medicines in patients with metastatic melanoma.

  4. Interleukin-like EMT inducer regulates partial phenotype switching in MITF-low melanoma cell lines

    PubMed Central

    Noguchi, Ken; Dalton, Annamarie C.; Howley, Breege V.; McCall, Buckley J.; Yoshida, Akihiro; Diehl, J. Alan

    2017-01-01

    ILEI (FAM3C) is a secreted factor that contributes to the epithelial-to-mesenchymal transition (EMT), a cell biological process that confers metastatic properties to a tumor cell. Initially, we found that ILEI mRNA is highly expressed in melanoma metastases but not in primary tumors, suggesting that ILEI contributes to the malignant properties of melanoma. While melanoma is not an epithelial cell-derived tumor and does not undergo a traditional EMT, melanoma undergoes a similar process known as phenotype switching in which high (micropthalmia-related transcription factor) MITF expressing (MITF-high) proliferative cells switch to a low expressing (MITF-low) invasive state. We observed that MITF-high proliferative cells express low levels of ILEI (ILEI-low) and MITF-low invasive cells express high levels of ILEI (ILEI-high). We found that inducing phenotype switching towards the MITF-low invasive state increases ILEI mRNA expression, whereas phenotype switching towards the MITF-high proliferative state decreases ILEI mRNA expression. Next, we used in vitro assays to show that knockdown of ILEI attenuates invasive potential but not MITF expression or chemoresistance. Finally, we used gene expression analysis to show that ILEI regulates several genes involved in the MITF-low invasive phenotype including JARID1B, HIF-2α, and BDNF. Gene set enrichment analysis suggested that ILEI-regulated genes are enriched for JUN signaling, a known regulator of the MITF-low invasive phenotype. In conclusion, we demonstrate that phenotype switching regulates ILEI expression, and that ILEI regulates partial phenotype switching in MITF-low melanoma cell lines. PMID:28545079

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

    PubMed

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

    2017-01-01

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

  6. PP2B and ERK1/2 regulate hyaluronan synthesis of HT168 and WM35 human melanoma cell lines.

    PubMed

    Katona, Éva; Juhász, Tamás; Somogyi, Csilla Szűcs; Hajdú, Tibor; Szász, Csaba; Rácz, Kálmán; Kókai, Endre; Gergely, Pál; Zákány, Róza

    2016-03-01

    Hyaluronan (HA) is the major glycosaminoglycan component of the extracellular matrix in either normal or malignant tissues and it may affect proliferation, motility and differentiation of various cell types. Three isoforms of plasma membrane-bound hyaluronan synthases (HAS 1, 2 and 3) secrete and simultaneously bind pericellular HA. HAS enzymes are subjects of post-translational protein phosphorylation which is believed to regulate their enzymatic activity. In this study, we investigated the HA homeostasis of normal human epidermal melanocytes, HT168 and WM35 human melanoma cell lines and melanoma metastases. HAS2 and HAS3 were detected in all the samples, while the expression of HAS1 was not detectable in any case. Malignant tissue samples and melanoma cell lines contained extra- and intracellular HA abundantly but not normal melanocytes. Applying HA as a chemoattractant facilitated the migration of melanoma cells in Boyden chamber. The amount of HA was reduced upon the inhibition of calcineurin with cyclosporine A (CsA), while the inhibition of ERK1/2 with PD098059 elevated it in both cell lines. The signals of Ser/Thr phosphoproteins at 57 kD were stronger after CsA treatment, while a markedly weaker signal was detected upon inhibition of the MAPK pathway. Our results suggest opposing effects of the two investigated enzymes on the HA homeostasis of melanoma cells. We propose that the dephosphorylation of HAS enzymes targeted by PP2B augments HA production, while their phosphorylation by the activity of MAPK pathway reduces HA synthesis. As the expression of the HA receptor RHAMM was also significantly enhanced by PD098059, the MAPK pathway exerted a complex attenuating effect on HA signalling in the investigated melanoma cells. This observation suggests that the application of MAPK-ERK pathway inhibitors requires a careful therapeutic design in melanoma treatment.

  7. PP2B and ERK1/2 regulate hyaluronan synthesis of HT168 and WM35 human melanoma cell lines

    PubMed Central

    KATONA, ÉVA; JUHÁSZ, TAMÁS; SOMOGYI, CSILLA SZŰCS; HAJDÚ, TIBOR; SZÁSZ, CSABA; RÁCZ, KÁLMÁN; KÓKAI, ENDRE; GERGELY, PÁL; ZÁKÁNY, RÓZA

    2016-01-01

    Hyaluronan (HA) is the major glycosaminoglycan component of the extracellular matrix in either normal or malignant tissues and it may affect proliferation, motility and differentiation of various cell types. Three isoforms of plasma membrane-bound hyaluronan synthases (HAS 1, 2 and 3) secrete and simultaneously bind pericellular HA. HAS enzymes are subjects of post-translational protein phosphorylation which is believed to regulate their enzymatic activity. In this study, we investigated the HA homeostasis of normal human epidermal melanocytes, HT168 and WM35 human melanoma cell lines and melanoma metastases. HAS2 and HAS3 were detected in all the samples, while the expression of HAS1 was not detectable in any case. Malignant tissue samples and melanoma cell lines contained extra- and intracellular HA abundantly but not normal melanocytes. Applying HA as a chemoattractant facilitated the migration of melanoma cells in Boyden chamber. The amount of HA was reduced upon the inhibition of calcineurin with cyclosporine A (CsA), while the inhibition of ERK1/2 with PD098059 elevated it in both cell lines. The signals of Ser/Thr phosphoproteins at 57 kD were stronger after CsA treatment, while a markedly weaker signal was detected upon inhibition of the MAPK pathway. Our results suggest opposing effects of the two investigated enzymes on the HA homeostasis of melanoma cells. We propose that the dephosphorylation of HAS enzymes targeted by PP2B augments HA production, while their phosphorylation by the activity of MAPK pathway reduces HA synthesis. As the expression of the HA receptor RHAMM was also significantly enhanced by PD098059, the MAPK pathway exerted a complex attenuating effect on HA signalling in the investigated melanoma cells. This observation suggests that the application of MAPK-ERK pathway inhibitors requires a careful therapeutic design in melanoma treatment. PMID:26717964

  8. Receptor-mediated cytotoxicity of alpha-MSH fragments containing melphalan in a human melanoma cell line.

    PubMed

    Morandini, R; Süli-Vargha, H; Libert, A; Loir, B; Botyánszki, J; Medzihradszky, K; Ghanem, G

    1994-01-02

    Four alpha-MSH drug conjugates have been synthesized, 2 C-terminal (Pep 3 and 4) and 2 central fragments (Pep 1 and 2), the latter being the 4-10 sequence known to be the main alpha-MSH-receptor-recognition site. Melphalan was introduced into each sequence at different locations. Their ability to recognize alpha-MSH receptors as well as their cytotoxic effects were compared in 3 cell lines: melanoma, carcinoma and fibroblast cells. Pep 1 and 2 were able to specifically bind to MSH receptors on melanoma cells by displacing labelled alpha-MSH from its binding sites at concentrations similar to the 4-10 heptapeptide sequence known to contain the main receptor-recognition site. They subsequently penetrate the cell, most probably by a receptor internalization mechanism, since about half of their effect could be inhibited by competition at the receptor level. Significant and selective cytotoxic effects to melanoma cells could be observed after only 2 hr exposure to the drug conjugates. Interestingly, these 2 conjugates, differing only in melphalan position, showed completely different cytotoxicity in terms of IC50 values, Pep 1 being 24 times more toxic to all cells; but the 2 were equally specific to melanoma cells. However, they both were less toxic to all cells than melphalan itself. Furthermore, Pep 1 and 2 were able to block the receptor and, unlike Pep 3 and 4, their cytotoxic effect could be significantly inhibited by an alpha-MSH agonist. Pep 3 and 4 were 5 to 10 times less toxic than melphalan to melanoma and carcinoma cells and 50 times less to fibroblast cells, and did not show any cell-type selectivity. They were less toxic than Pep 1 to melanoma and carcinoma cells by a factor of 2, but equally toxic to fibroblasts. In contrast, they were more toxic than Pep 2 to fibroblasts, melanoma and carcinoma by a factor of 3, 10 and 24 respectively. Our data strongly suggest a receptor-mediated cytotoxicity mechanism occurring with alpha-MSH central fragments in human

  9. Inhibition of p38 MAPK enhances ABT-737-induced cell death in melanoma cell lines: novel regulation of PUMA.

    PubMed

    Keuling, Angela M; Andrew, Susan E; Tron, Victor A

    2010-06-01

    The mitogen-activated protein kinase (MAPK) pathway is constitutively activated in the majority of melanomas, promoting cell survival, proliferation and migration. In addition, anti-apoptotic Bcl-2 family proteins Mcl-1, Bcl-xL and Bcl-2 are frequently overexpressed, contributing to melanoma's well-documented chemoresistance. Recently, it was reported that the combination of MAPK pathway inhibition by specific MEK inhibitors and Bcl-2 family inhibition by BH3-mimetic ABT-737 synergistically induces apoptotic cell death in melanoma cell lines. Here we provide the first evidence that inhibition of another key MAPK, p38, synergistically induces apoptosis in melanoma cells in combination with ABT-737. We also provide novel mechanistic data demonstrating that inhibition of p38 increases expression of pro-apoptotic Bcl-2 protein PUMA. Furthermore, we demonstrate that PUMA can be cleaved by a caspase-dependent mechanism during apoptosis and identify what appears to be the PUMA cleavage product. Thus, our findings suggest that the combination of ABT-737 and inhibition of p38 is a promising, new treatment strategy that acts through a novel PUMA-dependent mechanism.

  10. Nonlinear mixed effects dose response modeling in high throughput drug screens: application to melanoma cell line analysis.

    PubMed

    Ding, Kuan-Fu; Petricoin, Emanuel F; Finlay, Darren; Yin, Hongwei; Hendricks, William P D; Sereduk, Chris; Kiefer, Jeffrey; Sekulic, Aleksandar; LoRusso, Patricia M; Vuori, Kristiina; Trent, Jeffrey M; Schork, Nicholas J

    2018-01-12

    Cancer cell lines are often used in high throughput drug screens (HTS) to explore the relationship between cell line characteristics and responsiveness to different therapies. Many current analysis methods infer relationships by focusing on one aspect of cell line drug-specific dose-response curves (DRCs), the concentration causing 50% inhibition of a phenotypic endpoint (IC 50 ). Such methods may overlook DRC features and do not simultaneously leverage information about drug response patterns across cell lines, potentially increasing false positive and negative rates in drug response associations. We consider the application of two methods, each rooted in nonlinear mixed effects (NLME) models, that test the relationship relationships between estimated cell line DRCs and factors that might mitigate response. Both methods leverage estimation and testing techniques that consider the simultaneous analysis of different cell lines to draw inferences about any one cell line. One of the methods is designed to provide an omnibus test of the differences between cell line DRCs that is not focused on any one aspect of the DRC (such as the IC 50 value). We simulated different settings and compared the different methods on the simulated data. We also compared the proposed methods against traditional IC 50 -based methods using 40 melanoma cell lines whose transcriptomes, proteomes, and, importantly, BRAF and related mutation profiles were available. Ultimately, we find that the NLME-based methods are more robust, powerful and, for the omnibus test, more flexible, than traditional methods. Their application to the melanoma cell lines reveals insights into factors that may be clinically useful.

  11. BRAFV600E Negatively Regulates the AKT Pathway in Melanoma Cell Lines

    PubMed Central

    Chen, Brenden; Tardell, Christine; Higgins, Brian; Packman, Kathryn; Boylan, John F.; Niu, Huifeng

    2012-01-01

    Cross-feedback activation of MAPK and AKT pathways is implicated as a resistance mechanism for cancer therapeutic agents targeting either RAF/MEK or PI3K/AKT/mTOR. It is thus important to have a better understanding of the molecular resistance mechanisms to improve patient survival benefit from these agents. Here we show that BRAFV600E is a negative regulator of the AKT pathway. Expression of BRAFV600E in NIH3T3 cells significantly suppresses MEK inhibitor (RG7167) or mTORC1 inhibitor (rapamycin) induced AKT phosphorylation (pAKT) and downstream signal activation. Treatment-induced pAKT elevation is found in BRAF wild type melanoma cells but not in a subset of melanoma cell lines harboring BRAFV600E. Knock-down of BRAFV600E in these melanoma cells elevates basal pAKT and downstream signals, whereas knock-down of CRAF, MEK1/2 or ERK1/2 or treatment with a BRAF inhibitor have no impact on pAKT. Mechanistically, we show that BRAFV600E interacts with rictor complex (mTORC2) and regulates pAKT through mTORC2. BRAFV600E is identified in mTORC2 after immunoprecipitation of rictor. Knock-down of rictor abrogates BRAFV600E depletion induced pAKT. Knock-down of BRAFV600E enhances cellular enzyme activity of mTORC2. Aberrant activation of AKT pathway by PTEN loss appears to override the negative impact of BRAFV600E on pAKT. Taken together, our findings suggest that in a subset of BRAFV600E melanoma cells, BRAFV600E negatively regulates AKT pathway in a rictor-dependent, MEK/ERK and BRAF kinase-independent manner. Our study reveals a novel molecular mechanism underlying the regulation of feedback loops between the MAPK and AKT pathways. PMID:22880048

  12. BRAFV600E negatively regulates the AKT pathway in melanoma cell lines.

    PubMed

    Chen, Brenden; Tardell, Christine; Higgins, Brian; Packman, Kathryn; Boylan, John F; Niu, Huifeng

    2012-01-01

    Cross-feedback activation of MAPK and AKT pathways is implicated as a resistance mechanism for cancer therapeutic agents targeting either RAF/MEK or PI3K/AKT/mTOR. It is thus important to have a better understanding of the molecular resistance mechanisms to improve patient survival benefit from these agents. Here we show that BRAFV600E is a negative regulator of the AKT pathway. Expression of BRAFV600E in NIH3T3 cells significantly suppresses MEK inhibitor (RG7167) or mTORC1 inhibitor (rapamycin) induced AKT phosphorylation (pAKT) and downstream signal activation. Treatment-induced pAKT elevation is found in BRAF wild type melanoma cells but not in a subset of melanoma cell lines harboring BRAFV600E. Knock-down of BRAFV600E in these melanoma cells elevates basal pAKT and downstream signals, whereas knock-down of CRAF, MEK1/2 or ERK1/2 or treatment with a BRAF inhibitor have no impact on pAKT. Mechanistically, we show that BRAFV600E interacts with rictor complex (mTORC2) and regulates pAKT through mTORC2. BRAFV600E is identified in mTORC2 after immunoprecipitation of rictor. Knock-down of rictor abrogates BRAFV600E depletion induced pAKT. Knock-down of BRAFV600E enhances cellular enzyme activity of mTORC2. Aberrant activation of AKT pathway by PTEN loss appears to override the negative impact of BRAFV600E on pAKT. Taken together, our findings suggest that in a subset of BRAFV600E melanoma cells, BRAFV600E negatively regulates AKT pathway in a rictor-dependent, MEK/ERK and BRAF kinase-independent manner. Our study reveals a novel molecular mechanism underlying the regulation of feedback loops between the MAPK and AKT pathways.

  13. Genome-wide methylation sequencing of paired primary and metastatic cell lines identifies common DNA methylation changes and a role for EBF3 as a candidate epigenetic driver of melanoma metastasis

    PubMed Central

    Chatterjee, Aniruddha; Stockwell, Peter A; Ahn, Antonio; Rodger, Euan J; Leichter, Anna L; Eccles, Michael R

    2017-01-01

    Epigenetic alterations are increasingly implicated in metastasis, whereas very few genetic mutations have been identified as authentic drivers of cancer metastasis. Yet, to date, few studies have identified metastasis-related epigenetic drivers, in part because a framework for identifying driver epigenetic changes in metastasis has not been established. Using reduced representation bisulfite sequencing (RRBS), we mapped genome-wide DNA methylation patterns in three cutaneous primary and metastatic melanoma cell line pairs to identify metastasis-related epigenetic drivers. Globally, metastatic melanoma cell lines were hypomethylated compared to the matched primary melanoma cell lines. Using whole genome RRBS we identified 75 shared (10 hyper- and 65 hypomethylated) differentially methylated fragments (DMFs), which were associated with 68 genes showing significant methylation differences. One gene, Early B Cell Factor 3 (EBF3), exhibited promoter hypermethylation in metastatic cell lines, and was validated with bisulfite sequencing and in two publicly available independent melanoma cohorts (n = 40 and 458 melanomas, respectively). We found that hypermethylation of the EBF3 promoter was associated with increased EBF3 mRNA levels in metastatic melanomas and subsequent inhibition of DNA methylation reduced EBF3 expression. RNAi-mediated knockdown of EBF3 mRNA levels decreased proliferation, migration and invasion in primary and metastatic melanoma cell lines. Overall, we have identified numerous epigenetic changes characterising metastatic melanoma cell lines, including EBF3-induced aggressive phenotypic behaviour with elevated EBF3 expression in metastatic melanoma, suggesting that EBF3 promoter hypermethylation may be a candidate epigenetic driver of metastasis. PMID:28030832

  14. MITF suppression improves the sensitivity of melanoma cells to a BRAF inhibitor.

    PubMed

    Aida, Satoshi; Sonobe, Yukiko; Tanimura, Hiromi; Oikawa, Nobuhiro; Yuhki, Munehiro; Sakamoto, Hiroshi; Mizuno, Takakazu

    2017-11-28

    Microphthalmia-associated transcription factor (MITF) is expressed in melanomas and has a critical role in melanocyte development and transformation. Because inhibition of MITF inhibits cell growth in melanoma, MITF is a potential therapeutic target molecule. Here, we report the identification of CH6868398, which has a novel chemical structure and suppresses MITF expression at the protein level in melanoma cells. CH6868398 showed cell growth inhibition activity against MITF-dependent melanoma cells both with and without BRAF mutation and also exhibited anti-tumor efficacy in a melanoma xenograft model. Because selective BRAF inhibitors are standard therapeutics for BRAF-mutated melanoma, we investigated the effect of CH6868398 with a BRAF inhibitor, PLX4720, on cell growth inhibition. The addition of CH6868398 enhanced the cell growth inhibition activity of PLX4720 in melanoma cell lines. Furthermore, combination of CH6868398 and PLX4720 efficiently suppressed MITF protein and enhanced cleavage of Caspase3 and poly (ADP-ribose) polymerase (PARP) in melanoma cell lines. These data support the therapeutic potential of CH6868398 as an anti-melanoma agent that reduces MITF protein levels in combination with BRAF inhibitors. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    PubMed

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

    2005-07-01

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

  16. Multimarker Quantitative Real-Time PCR Detection of Circulating Melanoma Cells in Peripheral Blood: Relation to Disease Stage in Melanoma Patients

    PubMed Central

    Koyanagi, Kazuo; Kuo, Christine; Nakagawa, Taku; Mori, Takuji; Ueno, Hideaki; Lorico, Arnulfo R.; Wang, He-Jing; Hseuh, Eddie; O’Day, Steven J.; Hoon, Dave S.B.

    2010-01-01

    Background Detection of melanoma cells in circulation may be important in assessing tumor progression. The objective of this study was to develop a specific, reliable, multimarker quantitative real-time reverse transcription-PCR (qRT) assay for detecting melanoma cells in patients’ blood. Methods We developed qRT assays for the mRNA of four melanoma-associated markers: MART-1, GalNAc-T, PAX-3, and MAGE-A3. In optimization studies, we tested 17 melanoma cell lines and 49 peripheral blood leukocyte (PBL) samples from volunteers. We performed RNA and melanoma cell dilution studies to assess the detection limits and imprecision of the assays. We measured the mRNAs in blood specimens from 94 melanoma patients [American Joint Committee on Cancer (AJCC) stage I, n = 20; II, n = 20; III, n = 32; IV, n = 22]. Results All markers were frequently detected in melanoma cell lines, whereas none of the markers was detected in PBLs from volunteers. The qRT assay could detect 1 melanoma cell in 107 PBLs in the melanoma cell-dilution studies. Markers were detected in 15%, 30%, 75%, and 86% of melanoma patients with AJCC stage I, II, III, and IV disease, respectively. The number of positive markers and AJCC stage were significantly correlated (Spearman correlation coefficient = 0.58; P <0.0001). Conclusions Multimarker qRT can detect circulating melanoma cells in blood. Measurement of the studied molecular markers in blood may be useful in detection of metastasis and monitoring treatment response of melanoma patients. PMID:15817820

  17. DNA methylation and histone acetylation regulate the expression of MGMT and chemosensitivity to temozolomide in malignant melanoma cell lines.

    PubMed

    Chen, Ya-Ping; Hou, Xiao-Yang; Yang, Chun-Sheng; Jiang, Xiao-Xiao; Yang, Ming; Xu, Xi-Feng; Feng, Shou-Xin; Liu, Yan-Qun; Jiang, Guan

    2016-08-01

    Malignant melanoma is an aggressive, highly lethal dermatological malignancy. Chemoresistance and rapid metastasis limit the curative effect of multimodal therapies like surgery or chemotherapy. The suicide enzyme O6-methylguanine-DNA methyltransferase (MGMT) removes adducts from the O6-position of guanine to repair DNA damage. High MGMT expression is associated with resistance to therapy in melanoma. However, it is unknown if MGMT is regulated by DNA methylation or histone acetylation in melanoma. We examined the effects of the DNA methylation inhibitor 5-Aza-2'-deoxycytidine and histone deacetylase inhibitor Trichostatin A alone or in combination on MGMT expression and promoter methylation and histone acetylation in A375, MV3, and M14 melanoma cells. This study demonstrates that MGMT expression, CpG island methylation, and histone acetylation vary between melanoma cell lines. Combined treatment with 5-Aza-2'-deoxycytidine and Trichostatin A led to reexpression of MGMT, indicating that DNA methylation and histone deacetylation are associated with silencing of MGMT in melanoma. This study provides information on the role of epigenetic modifications in malignant melanoma that may enable the development of new strategies for treating malignant melanoma.

  18. Amino Acid Signature in Human Melanoma Cell Lines from Different Disease Stages.

    PubMed

    Wasinger, Christine; Hofer, Alexandra; Spadiut, Oliver; Hohenegger, Martin

    2018-04-19

    Cancer cells rewire metabolism to sustain high proliferation rates. Beside glycolysis and glutaminolysis, amino acids substitute as energy source, feed fatty acid biosynthesis and represent part of the secretome of transformed cells, including melanoma. We have therefore investigated acetate, pyruvate and the amino acid composition of the secretome of human melanoma cells representing the early slow (WM35, WM278, WM793b and VM21) and metastatic fast (A375, 518a2, 6F and WM8) growth phase in order to identify possible signalling components within these profiles. Proliferation assays and a principle component analysis revealed a stringent difference between the fast and slow growing melanoma cells. Moreover, upon inhibition of the mevalonate pathway, glutamic acid and alanine were identified as the central difference in the conditional media. A supplementation of the media with glutamic acid and the combination with alanine significantly accelerated the proliferation, migration and invasion of early stage melanoma cells, but not metastatic cells. Finally, the inhibition of the mevalonate pathway abolished the growth advantage of the melanoma cells in a time dependent manner. Taken together, these data corroborate a stage specific response in growth and aggressiveness to extracellular glutamic acid and alanine, indicative for microenvironmental signalling of individual amino acids.

  19. Tight Junction–Associated Signaling Pathways Modulate Cell Proliferation in Uveal Melanoma

    PubMed Central

    Jayagopal, Ashwath; Yang, Jin-Long; Haselton, Frederick R.; Chang, Min S.

    2011-01-01

    Purpose. To investigate the role of tight junction (TJ)–associated signaling pathways in the proliferation of uveal melanoma. Methods. Human uveal melanoma cell lines overexpressing the TJ molecule blood vessel epicardial substance (Bves) were generated. The effects of Bves overexpression on TJ protein expression, cell proliferation, and cell cycle distribution were quantified. In addition, localization and transcription activity of the TJ-associated protein ZO-1–associated nucleic acid binding protein (ZONAB) were evaluated using immunofluorescence and bioluminescence reporter assays to study the involvement of Bves signaling in cell proliferation-associated pathways. Results. Bves overexpression in uveal melanoma cell lines resulted in increased expression of the TJ proteins occludin and ZO-1, reduced cell proliferation, and increased sequestration of ZONAB at TJs and reduced ZONAB transcriptional activity. Conclusions. TJ proteins are present in uveal melanoma, and TJ-associated signaling pathways modulate cell signaling pathways relevant to proliferation in uveal melanoma. PMID:20861479

  20. Folate-conjugated immunoglobulin targets melanoma tumor cells for NK cell effector functions

    PubMed Central

    Skinner, Cassandra C.; McMichael, Elizabeth L.; Jaime-Ramirez, Alena C.; Abrams, Zachary B.; Lee, Robert J.; Carson, William E.

    2016-01-01

    The folate receptor (FR) is over-expressed on the vascular side of cancerous cells including those of the breast, ovaries, testes, and cervix. We hypothesized that a folate-conjugated immunoglobulin (F-IgG) would bind to the FR that is over-expressed on melanoma tumor cells to target these cells for lysis by natural killer (NK) cells. Folate receptor expression was confirmed in the Mel-39 (human melanoma) cell line by flow cytometry and immunoblot analysis, using KB (human oral epithelial) and F01 (human melanoma) as a positive and negative control, respectively. FR-positive and negative cell lines were treated with F-IgG or control immunoglobulin G (C-IgG) in the presence or absence of cytokines in order to determine NK cell ability to lyse FR-positive cell lines. NK cell activation was significantly upregulated and lysis of Mel 39 tumor cells enhanced following treatment with F-IgG, as compared to C-IgG at all effector:target (E:T) ratios (p<0.01). This trend was further enhanced by NK cell stimulation with the activating cytokine interleukin-12 (IL-12). NK cell production of cytokines such as interferon-gamma (IFN-γ), macrophage inflammatory protein 1 alpha (MIP-1α), and regulated on activation normal T-cell expressed and secreted (RANTES) were also significantly increased in response to co-stimulation with IL-12 stimulation and F-IgG-coated Mel 39 target cells, as compared to controls (p<0.01). In contrast, F-IgG did not bind to the FR-negative cell line F01 and had no significant effect on NK cell lysis or cytokine production. This research indicates the potential use of F-IgG for its ability to induce an immune response from NK cells against FR-positive melanoma tumor cells which can be further enhanced by the addition of cytokines. PMID:27035691

  1. Hypoxia-induced resistance to doxorubicin and methotrexate in human melanoma cell lines in vitro.

    PubMed

    Sanna, K; Rofstad, E K

    1994-07-15

    Rodent cell lines can develop resistance to doxorubicin and methotrexate during hypoxic stress. This has so far not been observed in human tumor cell lines. The purpose of our communication is to show that doxorubicin and methotrexate resistance can also develop in human melanoma cells during exposure to hypoxia. Four cell lines (BEX-c, COX-c, SAX-c, WIX-c) have been studied. Cells were exposed to hypoxia (O2 concentration < 10 ppm) for 24 hr prior to reoxygenation. Doxorubicin and methotrexate cell survival curves were determined immediately after as well as 18 and 42 hr after reoxygenation. The 4 cell lines were relatively sensitive to doxorubicin without hypoxia pre-treatment, and all developed resistance during exposure to hypoxia. Hypoxic stress also induced methotrexate resistance in BEX-c and SAX-c but not in COX-c and WIX-c. BEX-c and SAX-c were sensitive to methotrexate without hypoxia pre-treatment, whereas COX-c and WIX-c were resistant initially. Hypoxia-induced drug resistance was present immediately after reoxygenation and tended to decrease with time but remained statistically significant even 42 hr after reoxygenation.

  2. Antitumoral effect of vanadium compounds in malignant melanoma cell lines.

    PubMed

    Rozzo, Carla; Sanna, Daniele; Garribba, Eugenio; Serra, Maria; Cantara, Alessio; Palmieri, Giuseppe; Pisano, Marina

    2017-09-01

    In this study we evaluated the anticancer activity against malignant melanoma (MM) of four different vanadium species: the inorganic anion vanadate(V) (indicated with VN), and three oxidovanadium(IV) complexes, [V IV O(dhp) 2 ] where dhp - is the anion 1,2-dimethyl-3-hydroxy-4(1H)-pyridinonate (indicated with VS2), [V IV O(mpp) 2 ] where mpp - is 1-methyl-3-hydroxy-4(1H)-pyridinonate (indicated with VS3), and [V IV O(ppp) 2 ] where ppp - is 1-phenyl-2-methyl-3-hydroxy-4(1H)-pyridinonate (indicated with VS4). The antitumor effects of these compounds were studied against two different MM cell lines (A375 and CN-mel) and a fibroblast cell line (BJ) as normal control. All tested V compounds exert antiproliferative activity on MM cells in a dose dependent manner (IC 50 ranges from 2.4μM up to 14μM) being A375 the most sensitive cell line. VN and VS2 were the two most active compounds against A375 (IC 50 of 4.7 and 2.6μM, respectively), causing apoptosis and cell cycle block. The experimental data indicate that the cell cycle arrest occurs at different phases for the two V species analyzed (G2 checkpoint for VN and G0/G1 for VS2), showing the importance of the chemical form in determining their mechanism of action. These results add more insights into the landscape of vanadium versatility in biological systems and into its role as a potential cancer therapeutic agent. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Involvement of overexpressed wild-type BRAF in the growth of malignant melanoma cell lines.

    PubMed

    Tanami, Hideaki; Imoto, Issei; Hirasawa, Akira; Yuki, Yasuhiro; Sonoda, Itaru; Inoue, Jun; Yasui, Kohichiro; Misawa-Furihata, Akiko; Kawakami, Yutaka; Inazawa, Johji

    2004-11-18

    Comparative genomic hybridization (CGH) using 40 cell lines derived from malignant melanomas (MMs) revealed frequent amplification at 7q33-q34 containing BRAF gene, which often is mutated in MM. We found this gene to be amplified to a remarkable degree in the MM cell lines that exhibited high-level gains at 7q33-q34 in CGH. Among 40 cell lines, the eight lines that revealed neither BRAF nor NRAS mutations showed even higher levels of BRAF mRNA expression than the 32 mutated lines, although DNA amplification at 7q33-q34 was not detected in every lines overexpressing BRAF. MM cells that carried wild-type BRAF and NRAS showed constitutive overexpression of B-Raf protein and phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), even after serum starvation. Not only downregulation of the endogenously overexpressed wild-type B-Raf by antisense oligonucleotide but also a treatment with an inhibitor of mitogen-activated protein kinase kinase (MAPKK, MEK) reduced phosphorylated ERK1/2 and cell growth, whereas the exogenously expressed wild-type B-Raf promoted cell growth in MM cells. Our results provide the evidence that overexpression of wild-type B-Raf, in part but not always as a result of gene amplification, is one of the mechanisms underlying constitutive activation of the MAPK pathway that stimulates growth of MM cells.

  4. Notch3 signaling-mediated melanoma-endothelial crosstalk regulates melanoma stem-like cell homeostasis and niche morphogenesis.

    PubMed

    Hsu, Mei-Yu; Yang, Moon Hee; Schnegg, Caroline I; Hwang, Soonyean; Ryu, Byungwoo; Alani, Rhoda M

    2017-06-01

    Melanoma is among the most virulent cancers, owing to its propensity to metastasize and its resistance to current therapies. The treatment failure is largely attributed to tumor heterogeneity, particularly subpopulations possessing stem cell-like properties, ie, melanoma stem-like cells (MSLCs). Evidence indicates that the MSLC phenotype is malleable and may be acquired by non-MSLCs through phenotypic switching upon appropriate stimuli, the so-called 'dynamic stemness'. Since the phenotypic characteristics and functional integrity of MSLCs depend on their vascular niche, using a two-dimensional (2D) melanoma-endothelium co-culture model, where the MSLC niche is recapitulated in vitro, we identified Notch3 signaling pathway as a micro-environmental cue governing MSLC phenotypic plasticity via pathway-specific gene expression arrays. Accordingly, lentiviral shRNA-mediated Notch3 knockdown (KD) in melanoma cell lines exhibiting high levels of endogenous Notch3 led to retarded/abolished tumorigenicity in vivo through both depleting MSLC fractions, evinced by MSLC marker downregulation (eg, CD133 and CD271); and impeding the MSLC niche, corroborated by the attenuated tumor angiogenesis as well as vasculogenic mimicry. In contrast, Notch3 KD affected neither tumor growth nor MSLC subsets in a melanoma cell line with relatively low endogenous Notch3 expression. Thus, Notch3 signaling may facilitate MSLC plasticity and niche morphogenesis in a cell context-dependent manner. Our findings illustrate Notch3 as a molecular switch driving melanoma heterogeneity, and provide the biological rationale for Notch inhibition as a promising therapeutic option.

  5. An Inducible Endothelial Cell Surface Glycoprotein Mediates Melanoma Adhesion

    NASA Astrophysics Data System (ADS)

    Rice, G. Edgar; Bevilacqua, Michael P.

    1989-12-01

    Hematogenous metastasis requires the arrest and extravasation of blood-borne tumor cells, possibly involving direct adhesive interactions with vascular endothelium. Cytokine activation of cultured human endothelium increases adhesion of melanoma and carcinoma cell lines. An inducible 110-kD endothelial cell surface glycoprotein, designated INCAM-110, appears to mediate adhesion of melanoma cells. In addition, an inducible endothelial receptor for neutrophils, ELAM-1, supports the adhesion of a human colon carcinoma cell line. Thus, activation of vascular endothelium in vivo that results in increased expression of INCAM-110 and ELAM-1 may promote tumor cell adhesion and affect the incidence and distribution of metastases.

  6. Specific pesticide-dependent increases in α-synuclein levels in human neuroblastoma (SH-SY5Y) and melanoma (SK-MEL-2) cell lines.

    PubMed

    Chorfa, Areski; Bétemps, Dominique; Morignat, Eric; Lazizzera, Corinne; Hogeveen, Kevin; Andrieu, Thibault; Baron, Thierry

    2013-06-01

    Epidemiological studies indicate a role of genetic and environmental factors in Parkinson's disease involving alterations of the neuronal α-synuclein (α-syn) protein. In particular, a relationship between Parkinson's disease and occupational exposure to pesticides has been repeatedly suggested. Our objective was to precisely assess changes in α-syn levels in human neuroblastoma (SH-SY5Y) and melanoma (SK-MEL-2) cell lines following acute exposure to pesticides (rotenone, paraquat, maneb, and glyphosate) using Western blot and flow cytometry. These human cell lines express α-syn endogenously, and overexpression of α-syn (wild type or mutated A53T) can be obtained following recombinant adenoviral transduction. We found that endogenous α-syn levels in the SH-SY5Y neuroblastoma cell line were markedly increased by paraquat, and to a lesser extent by rotenone and maneb, but not by glyphosate. Rotenone also clearly increased endogenous α-syn levels in the SK-MEL-2 melanoma cell line. In the SH-SY5Y cell line, similar differences were observed in the α-syn adenovirus-transduced cells, with a higher increase of the A53T mutated protein. Paraquat markedly increased α-syn in the SK-MEL-2 adenovirus-transduced cell line, similarly for the wild-type or A53T proteins. The observed differences in the propensities of pesticides to increase α-syn levels are in agreement with numerous reports that indicate a potential role of exposure to certain pesticides in the development of Parkinson's disease. Our data support the hypothesis that pesticides can trigger some molecular events involved in this disease and also in malignant melanoma that consistently shows a significant but still unexplained association with Parkinson's disease.

  7. Regulation of cell cycle checkpoint kinase WEE1 by miR-195 in malignant melanoma.

    PubMed

    Bhattacharya, A; Schmitz, U; Wolkenhauer, O; Schönherr, M; Raatz, Y; Kunz, M

    2013-06-27

    WEE1 kinase has been described as a major gate keeper at the G2 cell cycle checkpoint and to be involved in tumour progression in different malignant tumours. Here we analysed the expression levels of WEE1 in a series of melanoma patient samples and melanoma cell lines using immunoblotting, quantitative real-time PCR and immunohistochemistry. WEE1 expression was significantly downregulated in patient samples of metastatic origin as compared with primary melanomas and in melanoma cell lines of high aggressiveness as compared with cell lines of low aggressiveness. Moreover, there was an inverse correlation between the expression of WEE1 and WEE1-targeting microRNA miR-195. Further analyses showed that transfection of melanoma cell lines with miR-195 indeed reduced WEE1 mRNA and protein expression in these cells. Reporter gene analysis confirmed direct targeting of the WEE1 3' untranslated region (3'UTR) by miR-195. Overexpression of miR-195 in SK-Mel-28 melanoma cells was accompanied by WEE1 reduction and significantly reduced stress-induced G2-M cell cycle arrest, which could be restored by stable overexpression of WEE1. Moreover, miR-195 overexpression and WEE1 knockdown, respectively, increased melanoma cell proliferation. miR-195 overexpression also enhanced migration and invasiveness of melanoma cells. Taken together, the present study shows that WEE1 expression in malignant melanoma is directly regulated by miR-195. miR-195-mediated downregulation of WEE1 in metastatic lesions may help to overcome cell cycle arrest under stress conditions in the local tissue microenvironment to allow unrestricted growth of tumour cells.

  8. Notch3 Signaling-Mediated Melanoma-Endothelial Crosstalk Regulates Melanoma Stem-Like Cell Homeostasis and Niche Morphogenesis

    PubMed Central

    Hsu, Mei-Yu; Yang, Moon Hee; Schnegg, Caroline I.; Hwang, Soonyean; Ryu, Byungwoo; Alani, Rhoda M.

    2016-01-01

    Melanoma is among the most virulent cancers, owing to its propensity to metastasize and its resistance to current therapies. The treatment failure is largely attributed to tumor heterogeneity, particularly subpopulations possessing stem cell-like properties, i.e., melanoma stem-like cells (MSLCs). Evidence indicates that the MSLC phenotype is malleable and may be acquired by non-MSLCs through phenotypic switching upon appropriate stimuli, the so–called “dynamic stemness”. Since the phenotypic characteristics and functional integrity of MSLCs depend on their vascular niche, using a two dimensional (2D) melanoma-endothelium co-culture model, where the MSLC niche is recapitulated in vitro, we identified Notch3 signaling pathway as a micro-environmental cue governing MSLC phenotypic plasticity via pathway-specific gene expression arrays. Accordingly, lentiviral shRNA-mediated Notch3 knockdown (KD) in melanoma cell lines exhibiting high levels of endogenous Notch3 led to retarded/abolished tumorigenicity in vivo through both depleting MSLC fractions, evinced by MSLC marker down-regulation (e.g., CD133 and CD271); and impeding the MSLC niche, corroborated by the attenuated tumor angiogenesis as well as vasculogenic mimicry. In contrast, Notch3 KD affected neither tumor growth nor MSLC subsets in a melanoma cell line with relatively low endogenous Notch3 expression. Thus, Notch3 signaling may facilitate MSLC plasticity and niche morphogenesis in a cell context-dependent fashion. Our findings illustrate Notch3 as a molecular switch driving melanoma heterogeneity, and provide the biological rationale for Notch inhibition as a promising therapeutic option. PMID:28165469

  9. Protein disulfide isomerases: Impact of thapsigargin treatment on their expression in melanoma cell lines.

    PubMed

    Silva, Zélia; Veríssimo, Teresa; Videira, Paula A; Novo, Carlos

    2015-08-01

    Anti-cancer treatments usually elevate the content of unfolded or misfolded proteins in the endoplasmic reticulum (ER). Here we aimed to get insights into the relation between sensitivity of melanoma cell lines to the ER stress inducer thapsigargin (THG) and the genetic expression of protein disulfide isomerase family members (PDIs). The expression of PDIs was analysed by flow cytometry and real-time PCR. The results showed that SK-MEL-30, the less THG sensitive cell line, displays higher basal PDIs' expression levels and the sensitivity is increased by the PDIs inhibitor bacitracin. While SK-MEL-30 PDIs' expression is not THG dose-dependent, an increase in glucose related protein 78 (GRP78), PDIA5, PDIA6, and thioredoxin-related-transmembrane proteins' (TMX3 and TMX4) expression, in response to higher drug concentrations, was observed in MNT-1. The differences in PDIs' gene expression in MNT-1 suggest a different response to ER stress compared to the other cell lines and highlight the importance of understanding the diversity among cancer cells. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Effects of combined treatment with interferon and mezerein on melanogenesis and growth in human melanoma cells.

    PubMed

    Fisher, P B; Prignoli, D R; Hermo, H; Weinstein, I B; Pestka, S

    1985-01-01

    We have analyzed the effects of various human interferons produced in bacteria and the antileukemic compound mezerein (MEZ) on growth and melanogenesis in human melanoma cells. In four human melanoma cell lines, recombinant human fibroblast interferon (IFN-beta) was more active than recombinant human leukocyte interferons (IFN-alpha A, IFN-alpha D, or IFN-alpha A/D (Bgl] in inhibiting cellular proliferation. When monolayer cultures were exposed to 1000 IU/ml IFN-beta for four days the degree of growth inhibition in the different melanoma cell lines varied between 94 and 26%. Similarly, four days growth in medium containing 10 ng/ml MEZ resulted in either no inhibition of growth or as much as 53% inhibition of growth, depending on the specific melanoma cell line tested. MEZ induced dendrite-like processes, cytoplasmic projections morphologically similar to those normally found in neurons and melanocytes, in all four melanoma cell lines, whereas none of the interferons tested had this effect. The combination of interferon and MEZ resulted in a dramatic inhibition in cellular proliferation in all four melanoma cell lines. When cell extracts were assayed for melanin content, a marker of melanoma cell differentiation, the combination of IFN-beta and MEZ resulted in higher levels of melanin than with either agent alone. Dendrite-like formation was also prominent in the cultures treated with this combination. These results indicate that the antiproliferative effect of interferon toward human melanoma dells can be enhanced by treatment with MEZ and that this effect is associated with an enhancement of terminal differentiation.

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

    PubMed

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

    2015-02-01

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

  12. MiR-767 promoted cell proliferation in human melanoma by suppressing CYLD expression.

    PubMed

    Zhang, Kejin; Guo, Ling

    2018-01-30

    MicroRNAs (miRNAs) have emerged as critical regulators for cancer development and progression of human melanoma. However, the potential molecular mechanism of miR-767 in human melanoma has not been intensively investigated. In this present study, we confirmed that miR-767 was frequently up-regulated in human melanoma tissues and cell lines. Ectopic expression of miR-767 promoted cell proliferation in human melanoma cell lines A375 and WM35, whereas miR-767-in reversed the function. Bioinformatics analysis revealed that cylindromatosis (CYLD) was hypothesized to be a possible target gene of miR-767, and this was confirmed by luciferase activity assay. Knockdown of CYLD counteracted the proliferation arrest by miR-767-in in melanoma cells A375 and WM35. In conclusion, our study indicated that miR-767 acted as a role of tumor promoter by targeting CYLD in human melanoma, and might serve as a prognostic or therapeutic target for human melanoma. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Blue light inhibits proliferation of melanoma cells

    NASA Astrophysics Data System (ADS)

    Becker, Anja; Distler, Elisabeth; Klapczynski, Anna; Arpino, Fabiola; Kuch, Natalia; Simon-Keller, Katja; Sticht, Carsten; van Abeelen, Frank A.; Gretz, Norbert; Oversluizen, Gerrit

    2016-03-01

    Photobiomodulation with blue light is used for several treatment paradigms such as neonatal jaundice, psoriasis and back pain. However, little is known about possible side effects concerning melanoma cells in the skin. The aim of this study was to assess the safety of blue LED irradiation with respect to proliferation of melanoma cells. For that purpose we used the human malignant melanoma cell line SK-MEL28. Cell proliferation was decreased in blue light irradiated cells where the effect size depended on light irradiation dosage. Furthermore, with a repeated irradiation of the melanoma cells on two consecutive days the effect could be intensified. Fluorescence-activated cell sorting with Annexin V and Propidium iodide labeling did not show a higher number of dead cells after blue light irradiation compared to non-irradiated cells. Gene expression analysis revealed down-regulated genes in pathways connected to anti-inflammatory response, like B cell signaling and phagosome. Most prominent pathways with up-regulation of genes were cytochrome P450, steroid hormone biosynthesis. Furthermore, even though cells showed a decrease in proliferation, genes connected to the cell cycle were up-regulated after 24h. This result is concordant with XTT test 48h after irradiation, where irradiated cells showed the same proliferation as the no light negative control. In summary, proliferation of melanoma cells can be decreased using blue light irradiation. Nevertheless, the gene expression analysis has to be further evaluated and more studies, such as in-vivo experiments, are warranted to further assess the safety of blue light treatment.

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

    PubMed

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

    1997-10-01

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

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

    PubMed

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

    2016-08-01

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

  16. Morphological changes in human melanoma cells following irradiation with thermal neutrons.

    PubMed

    Barkla, D H; Allen, B J; Brown, J K; Mountford, M; Mishima, Y; Ichihashi, M

    1989-01-01

    Morphological changes in two human melanoma cell lines, MM96 and MM418, following irradiation with thermal neutrons, were studied using light and electron microscopy. The results show that the response of human malignant melanoma cells to neutron irradiation is both cell line dependent and dose dependent, and that in any given cell line, some cells are more resistant to irradiation than others, thus demonstrating heterogeneity in respect to radiosensitivity. Cells repopulating MM96 flasks after irradiation were morphologically similar to the cells of origin whereas in MM418 flasks cells differentiated into five morphologically distinct subgroups and showed increased melanization. The results also show that radiation causes distinctive morphological patterns of damage although ultrastructural changes unique to the high LET particles released from boron 10 neutron capture are yet to be identified.

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

    PubMed

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

    1996-12-01

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

  18. Stem cell media culture of melanoma results in the induction of a nonrepresentative neural expression profile.

    PubMed

    Anaka, Matthew; Freyer, Claudia; Gedye, Craig; Caballero, Otavia; Davis, Ian D; Behren, Andreas; Cebon, Jonathan

    2012-02-01

    The ability of cell lines to accurately represent cancer is a major concern in preclinical research. Culture of glioma cells as neurospheres in stem cell media (SCM) has been shown to better represent the genotype and phenotype of primary glioblastoma in comparison to serum cell lines. Despite the use of neurosphere-like models of many malignancies, there has been no robust analysis of whether other cancers benefit from a more representative phenotype and genotype when cultured in SCM. We analyzed the growth properties, transcriptional profile, and genotype of melanoma cells grown de novo in SCM, as while melanocytes share a common precursor with neural cells, melanoma frequently demonstrates divergent behavior in cancer stem cell assays. SCM culture of melanoma cells induced a neural lineage gene expression profile that was not representative of matched patient tissue samples and which could be induced in serum cell lines by switching them into SCM. There was no enrichment for expression of putative melanoma stem cell markers, but the SCM expression profile did overlap significantly with that of SCM cultures of glioma, suggesting that the observed phenotype is media-specific rather than melanoma-specific. Xenografts derived from either culture condition provided the best representation of melanoma in situ. Finally, SCM culture of melanoma did not prevent ongoing acquisition of DNA copy number abnormalities. In conclusion, SCM culture of melanoma does not provide a better representation of the phenotype or genotype of metastatic melanoma, and the resulting neural bias could potentially confound therapeutic target identification. Copyright © 2011 AlphaMed Press.

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

    PubMed

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

    2011-06-01

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

  20. In vitro anticancer activity of Betulinic acid and derivatives thereof on equine melanoma cell lines from grey horses and in vivo safety assessment of the compound NVX-207 in two horses.

    PubMed

    Liebscher, G; Vanchangiri, K; Mueller, Th; Feige, K; Cavalleri, J-M V; Paschke, R

    2016-02-25

    Betulinic acid, a pentacyclic triterpene, and its derivatives are promising compounds for cancer treatment in humans. Melanoma is not only a problem for humans but also for grey horses as they have a high potential of developing melanoma lesions coupled to the mutation causing their phenotype. Current chemotherapeutic treatment carries the risk of adverse health effects for the horse owner or the treating veterinarian by exposure to antineoplastic compounds. Most treatments have low prospects for systemic tumor regression. Thus, a new therapy is needed. In this in vitro study, Betulinic acid and its two derivatives B10 and NVX-207, both with an improved water solubility compared to Betulinic acid, were tested on two equine melanoma cell lines (MelDuWi and MellJess/HoMelZh) and human melanoma (A375) cell line. We could demonstrate that all three compounds especially NVX-207 show high cytotoxicity on both equine melanoma cell lines. The treatment with these compounds lead to externalization of phosphatidylserines on the cell membrane (AnnexinV-staining), DNA-fragmentation (cell cycle analysis) and activation of initiator and effector caspases (Caspase assays). Our results indicate that the apoptosis is induced in the equine melanoma cells by all three compounds. Furthermore, we succeed in encapsulating the most active compound NVX-207 in 2-Hydroxyprolyl-β-cyclodextrine without a loss of its activity. This formulation can be used as a promising antitumor agent for treating grey horse melanoma. In a first tolerability evaluation in vivo the formulation was administered every one week for 19 consecutive weeks and well tolerated in two adult melanoma affected horses. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  1. Norepinephrine upregulates VEGF, IL-8, and IL-6 expression in human melanoma tumor cell lines: implications for stress-related enhancement of tumor progression

    PubMed Central

    Yang, Eric V.; Kim, Seung-jae; Donovan, Elise L.; Chen, Min; Gross, Amy C.; Webster Marketon, Jeanette I.; Barsky, Sanford H.; Glaser, Ronald

    2009-01-01

    Studies suggest that stress can be a co-factor for the initiation and progression of cancer. The catecholamine stress hormone, norepinephrine (NE), may influence tumor progression by modulating the expression of factors implicated in angiogenesis and metastasis. The goal of this study was to examine the influence of NE on the expression of VEGF, IL-8, and IL-6 by the human melanoma cell lines, C8161, 1174MEL, and Me18105. Cells were treated with NE and levels of VEGF, IL-8, and IL-6 were measured using ELISA and real-time PCR. The expression of β-adrenergic receptors (β-ARs) mRNA and protein were also assessed. Finally, immunohistochemitry was utilized to examine the presence of β1- and β2-AR in primary and metastatic human melanoma biopsies. We show that NE treatment upregulated production of VEGF, IL-8, and IL-6 in C8161 cells and to a lesser extent 1174MEL and Me18105 cells. The upregulation was associated with induced gene expression. The effect on C8161 cells was mediated by both β1- and β2-ARs. Furthermore, 18 of 20 melanoma biopsies examined expressed β2-AR while 14 of 20 melanoma biopsies expressed β1-AR. Our data support the hypothesis that NE can stimulate the aggressive potential of melanoma tumor cells, in part, by inducing the production VEGF, IL-8, and IL-6. This line of research further suggests that interventions targeting components of the activated sympathetic-adrenal medullary (SAM) axis, or the utilization of β-AR blocking agents, may represent new strategies for slowing down the progression of malignant disease and improving cancer patients’ quality of life. PMID:18996182

  2. Targeting melanoma stem cells with the Vitamin E derivative δ-tocotrienol.

    PubMed

    Marzagalli, Monica; Moretti, Roberta Manuela; Messi, Elio; Marelli, Marina Montagnani; Fontana, Fabrizio; Anastasia, Alessia; Bani, Maria Rosa; Beretta, Giangiacomo; Limonta, Patrizia

    2018-01-12

    The prognosis of metastatic melanoma is very poor, due to the development of drug resistance. Cancer stem cells (CSCs) may play a crucial role in this mechanism, contributing to disease relapse. We first characterized CSCs in melanoma cell lines. We observed that A375 (but not BLM) cells are able to form melanospheres and show CSCs traits: expression of the pluripotency markers SOX2 and KLF4, higher invasiveness and tumor formation capability in vivo with respect to parental adherent cells. We also showed that a subpopulation of autofluorescent cells expressing the ABCG2 stem cell marker is present in the A375 spheroid culture. Based on these data, we investigated whether δ-TT might target melanoma CSCs. We demonstrated that melanoma cells escaping the antitumor activity of δ-TT are completely devoid of the ability to form melanospheres. In contrast, cells that escaped vemurafenib treatment show a higher ability to form melanospheres than control cells. δ-TT also induced disaggregation of A375 melanospheres and reduced the spheroidogenic ability of sphere-derived cells, reducing the expression of the ABCG2 marker. These data demonstrate that δ-TT exerts its antitumor activity by targeting the CSC subpopulation of A375 melanoma cells and might represent a novel chemopreventive/therapeutic strategy against melanoma.

  3. Fisetin induces apoptosis through mitochondrial apoptosis pathway in human uveal melanoma cells.

    PubMed

    Wang, Kai; Hu, Dan-Ning; Lin, Hui-Wen; Yang, Wei-En; Hsieh, Yi-Hsien; Chien, Hsiang-Wen; Yang, Shun-Fa

    2018-05-01

    Fisetin, a diatery flavonoid, been reported that possess anticancer effects in various cancers. The purpose of the study was to investigate the antitumor effects of fisetin in cultured uveal melanoma cell lines and compared with normal retinal pigment epithelial (RPE) cells. MTT assay was used for evaluating cytotoxic effects of fisetin. Flow cytometry study was used for the determination of apoptosis. JC-1 fluorescent reader was used to determine mitochondrial transmembrane potential changes. The results shown that fisetin dose-dependently decreased the cell viability of uveal melanoma cells but not influenced the cell viability of RPE cells. Apoptosis of uveal melanoma cells was induced by fisetin efficiently. Fisetin inhibited antiapoptotic Bcl-2 family proteins and damaged the mitochondrial transmembrane potential. The levels of proapoptotic Bcl-2 proteins, cytochrome c, and various caspase activities were increased by fisetin. In conclusion, fisetin induces apoptosis of uveal melanoma cells selectively and may be a promising agent to be explored for the treatment of uveal melanoma. © 2018 Wiley Periodicals, Inc.

  4. Accumulation of prohibitin is a common cellular response to different stressing stimuli and protects melanoma cells from ER stress and chemotherapy-induced cell death

    PubMed Central

    Tortelli, Tharcisio Citrangulo; de Godoy, Lyris Martins Franco; de Souza, Gustavo Antonio; Bonatto, Diego; Otake, Andreia Hanada; de Freitas Saito, Renata; Rosa, Jose Cesar; Greene, Lewis Joel; Chammas, Roger

    2017-01-01

    Melanoma is responsible for most deaths among skin cancers and conventional and palliative care chemotherapy are limited due to the development of chemoresistance. We used proteomic analysis to identify cellular responses that lead to chemoresistance of human melanoma cell lines to cisplatin. A systems approach to the proteomic data indicated the participation of specific cellular processes such as oxidative phosphorylation, mitochondrial organization and homeostasis, as well as the unfolded protein response (UPR) to be required for the survival of cells treated with cisplatin. Prohibitin (PHB) was among the proteins consistently accumulated, interacting with the functional clusters associated with resistance to cisplatin. We showed PHB accumulated at different levels in melanoma cell lines under stressing stimuli, such as (i) treatment with temozolomide (TMZ), dacarbazine (DTIC) and cisplatin; (ii) serum deprivation; (iii) tunicamycin, an UPR inducer. Prohibitin accumulated in the mitochondria of melanoma cells after cisplatin and tunicamycin treatment and its de novo accumulation led to chemoresistance melanoma cell lines. In contrast, PHB knock-down sensitized melanoma cells to cisplatin and tunicamycin treatment. We conclude that PHB participates in the survival of cells exposed to different stress stimuli, and can therefore serve as a target for the sensitization of melanoma cells to chemotherapy. PMID:28562344

  5. Selective sensitivity to wasabi-derived 6-(methylsulfinyl)hexyl isothiocyanate of human breast cancer and melanoma cell lines studied in vitro.

    PubMed

    Nomura, Takahiro; Shinoda, Shoko; Yamori, Takao; Sawaki, Saeko; Nagata, Ikuko; Ryoyama, Kazuo; Fuke, Yoko

    2005-01-01

    Recently, attention has focused on the anticancer properties of an aromatic component 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) in a typical Japanese spice, wasabi. In this paper, anticancer activity of 6-MITC in vitro was studied by using a human cancer cell (HCC) panel. 6-MITC directly affected the cells in the HCC panel and inhibited their growth in culture. The mean concentration required to inhibit 50% of control cell growth was 3.9 microM, which is a sufficiently low dosage for practical use. The suppression influenced not only the cell growth, but also the survival of these cells. The mean concentration to suppress cells to a 50% survival was 43.7 microM. The reduction activity of 6-MITC was differential, and it suppressed specific cells. These severely suppressed cell lines included breast cancer and melanoma cell lines. For example, one melanoma line was seriously damaged at a concentration of 0.3 microM of 6-MITC. Compared with other MITCs (2-MITC, 4-MITC and 8-MITC), 6-MITC showed the most effective suppression and with the most specific manner of the cells mentioned above. A "COMPARE" analysis using a computerized algorithm, which was based on the HCC database, suggested that the suppression mechanism of 6-MITC is unique and may be different from that of other known chemicals. The actual mechanism may not a simple one but may involve multiple pathways. On account of its sufficiently small size, 6-MITC is a new possible candidate for controlling cancer cells.

  6. Biosynthesis of N,N-dimethyltryptamine (DMT) in a melanoma cell line and its metabolization by peroxidases.

    PubMed

    Gomes, Melissa M; Coimbra, Janine B; Clara, Renan O; Dörr, Felipe A; Moreno, Ana Carolina R; Chagas, Jair R; Tufik, Sérgio; Pinto, Ernani; Catalani, Luiz H; Campa, Ana

    2014-04-01

    Tryptophan (TRP) is essential for many physiological processes, and its metabolism changes in some diseases such as infection and cancer. The most studied aspects of TRP metabolism are the kynurenine and serotonin pathways. A minor metabolic route, tryptamine and N,N-dimethyltryptamine (DMT) biosynthesis, has received far less attention, probably because of the very low amounts of these compounds detected only in some tissues, which has led them to be collectively considered as trace amines. In a previous study, we showed a metabolic interrelationship for TRP in melanoma cell lines. Here, we identified DMT and N,N-dimethyl-N-formyl-kynuramine (DMFK) in the supernatant of cultured SK-Mel-147 cells. Furthermore, when we added DMT to the cell culture, we found hydroxy-DMT (OH-DMT) and indole acetic acid (IAA) in the cell supernatant at 24 h. We found that SK-Mel-147 cells expressed mRNA for myeloperoxidase (MPO) and also had peroxidase activity. We further found that DMT oxidation was catalyzed by peroxidases. DMT oxidation by horseradish peroxidase, H2O2 and MPO from PMA-activated neutrophils produced DMFK, N,N-dimethyl-kynuramine (DMK) and OH-DMT. Oxidation of DMT by peroxidases apparently uses the common peroxidase cycle involving the native enzyme, compound I and compound II. In conclusion, this study describes a possible alternative metabolic pathway for DMT involving peroxidases that has not previously been described in humans and identifies DMT and metabolites in a melanoma cell line. The extension of these findings to other cell types and the biological effects of DMT and its metabolites on cell proliferation and function are key questions for future studies. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Hyaluronan synthase 3 (HAS3) overexpression downregulates MV3 melanoma cell proliferation, migration and adhesion

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

    Takabe, Piia, E-mail: piia.takabe@uef.fi; Bart, Geneviève; Ropponen, Antti

    2015-09-10

    Malignant skin melanoma is one of the most deadly human cancers. Extracellular matrix (ECM) influences the growth of malignant tumors by modulating tumor cells adhesion and migration. Hyaluronan is an essential component of the ECM, and its amount is altered in many tumors, suggesting an important role for hyaluronan in tumorigenesis. Nonetheless its role in melanomagenesis is not understood. In this study we produced a MV3 melanoma cell line with inducible expression of the hyaluronan synthase 3 (HAS3) and studied its effect on the behavior of the melanoma cells. HAS3 overexpression expanded the cell surface hyaluronan coat and decreased melanomamore » cell adhesion, migration and proliferation by cell cycle arrest at G1/G0. Melanoma cell migration was restored by removal of cell surface hyaluronan by Streptomyces hyaluronidase and by receptor blocking with hyaluronan oligosaccharides, while the effect on cell proliferation was receptor independent. Overexpression of HAS3 decreased ERK1/2 phosphorylation suggesting that inhibition of MAP-kinase signaling was responsible for these suppressive effects on the malignant phenotype of MV3 melanoma cells. - Highlights: • Inducible HAS3-MV3 melanoma cell line was generated using Lentiviral transduction. • HAS3 overexpression inhibits MV3 cell migration via hyaluronan–receptor interaction. • HAS3 overexpression decreases MV3 melanoma cell proliferation and adhesion. • ERK1/2 phosphorylation is downregulated by 50% in HAS3 overexpressing cells. • The results suggest that hyaluronan has anti-cancer like effects in melanoma.« less

  8. Impact of BRAF kinase inhibitors on the miRNomes and transcriptomes of melanoma cells.

    PubMed

    Kozar, Ines; Cesi, Giulia; Margue, Christiane; Philippidou, Demetra; Kreis, Stephanie

    2017-11-01

    Melanoma is an aggressive skin cancer with increasing incidence worldwide. The development of BRAF kinase inhibitors as targeted treatments for patients with BRAF-mutant tumours contributed profoundly to an improved overall survival of patients with metastatic melanoma. Despite these promising results, the emergence of rapid resistance to targeted therapy remains a serious clinical issue. To investigate the impact of BRAF inhibitors on miRNomes and transcriptomes, we used in vitro melanoma models consisting of BRAF inhibitor-sensitive and -resistant cell lines generated in our laboratory. Subsequently, microarray analyses were performed followed by RT-qPCR validations. Regarding miRNome and transcriptome changes, the long-term effects of BRAF inhibition differed in a cell line-specific manner with the two different BRAF inhibitors inducing comparable responses in three melanoma cell lines. Despite this heterogeneity, several miRNAs (e.g. miR-92a-1-5p, miR-708-5p) and genes (e.g. DOK5, PCSK2) were distinctly differentially expressed in drug-resistant versus -sensitive cell lines. Analyses of coexpressed miRNAs, as well as inversely correlated miRNA-mRNA pairs, revealed a low MITF/AXL ratio in two drug-resistant cell lines that might be regulated by miRNAs. Several genes and miRNAs were differentially regulated in the drug-resistant and -sensitive cell lines and might be considered as prognostic and/or diagnostic resistance biomarkers in melanoma drug resistance. Thus far, only little information is available on the significance and role of miRNAs with respect to kinase inhibitor treatments and emergence of drug resistance. In this study, promising miRNAs and genes were identified and associated to BRAF inhibitor-mediated resistance in melanoma. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Methotrexate inhibits the viability of human melanoma cell lines and enhances Fas/Fas-ligand expression, apoptosis and response to interferon-alpha: Rationale for its use in combination therapy

    PubMed Central

    Nihal, Minakshi; Wu, Jianqiang; Wood, Gary S.

    2015-01-01

    Melanoma, a highly aggressive form of cancer, is notoriously resistant to available therapies. Methotrexate (MTX), an antifolate, competitively inhibits DNA synthesis and is effective for several types of cancer. In cutaneous T-cell lymphoma (CTCL), MTX increases Fas death receptor by decreasing Fas promoter methylation by blocking the synthesis of SAM, the principal methyl donor for DNMTs, resulting in enhanced Fas-mediated apoptosis. The objective of this study was to explore the effects of MTX in human melanoma. MTX variably inhibited the survival of melanoma cells and induced apoptosis as evident by annexin V positivity and senescence associated β-galactosidase activity induction. Furthermore, MTX caused increased transcript and protein levels of extrinsic apoptotic pathway factors Fas and Fas-ligand, albeit at different levels in different cell lines. Our pyrosequencing studies showed that this increased expression of Fas was associated with Fas promoter demethylation. Overall, the ability of MTX to up-regulate Fas/FasL and enhance melanoma apoptosis through extrinsic as well as intrinsic pathways might make it a useful component of novel combination therapies designed to affect multiple melanoma targets simultaneously. In support of this concept, combination therapy with MTX and interferon-alpha (IFNα) induced significantly greater apoptosis in the aggressive A375 cell line than either agent alone. PMID:24862567

  10. Dacarbazine inhibits proliferation of melanoma FEMX-1 cells by up-regulating expression of miRNA-200.

    PubMed

    Chen, Y-N

    2017-03-01

    Melanoma is a highly aggressive tumour, and treatment efficacy depends on the stage of the tumour. Early stage cutaneous melanoma is efficiently treated by surgical excision. In contrast, late-stage melanoma requires chemotherapy with dacarbazine (DTIC). Unfortunately, advanced melanoma can often be resistant to DTIC. The mechanisms of anti-melanoma effects of DTIC are still poorly understood, which hinders development of more potent therapies. In this study, we examined the effects of DTIC on growth inhibition of FEMX-1 melanoma cell line, expression of apoptosis-related proteins, and expression of micro (mi)RNA-200 (miRNA-200a, miRNA-200b, miRNA-200c, and miRNA-141). DTIC was used at 50 (low dose) or 100 (high dose) mg/ml. Cell growth inhibition was documented by MTT assay. Cell apoptosis was quantified by propidium iodide staining and caspase 3-8 activity assay. Expression of apoptosis-related proteins Bim, Bak, BAX, and Bad were documented by Western blot analysis, while expression of miRNA-200 by PCR. DTIC dose-dependently inhibited growth of FEMX-1 melanoma cell line, induced cell apoptosis, modulated the levels of apoptosis-related proteins, and up-regulated expression of miRNA-200 family members. DTIC inhibits the growth of melanoma cells by up-regulating expression of miRNA-200.

  11. Imidazopyridine-fused [1,3]-diazepinones part 2: Structure-activity relationships and antiproliferative activity against melanoma cells.

    PubMed

    Bellet, Virginie; Lichon, Laure; Arama, Dominique P; Gallud, Audrey; Lisowski, Vincent; Maillard, Ludovic T; Garcia, Marcel; Martinez, Jean; Masurier, Nicolas

    2017-01-05

    We recently described a pyrido-imidazodiazepinone derivative which could be a promising hit compound for the development of new drugs acting against melanoma cells. In this study, a series of 28 novel pyrido-imidazodiazepinones were synthesized and screened for their in vitro cytotoxic activities against the melanoma MDA-MB-435 cell line. Among the derivatives, seven of them showed 50% growth inhibitory activity at 1 μM concentration, and high selectivity against the melanoma cell line MDA-MB-435. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  12. MITF and PAX3 Play Distinct Roles in Melanoma Cell Migration; Outline of a "Genetic Switch" Theory Involving MITF and PAX3 in Proliferative and Invasive Phenotypes of Melanoma.

    PubMed

    Eccles, Michael R; He, Shujie; Ahn, Antonio; Slobbe, Lynn J; Jeffs, Aaron R; Yoon, Han-Seung; Baguley, Bruce C

    2013-09-11

    Melanoma is a very aggressive neoplasm with a propensity to undergo progression and invasion early in its evolution. The molecular pathways underpinning invasion in melanoma are now just beginning to be elucidated, but a clear understanding of the transition from non-invasive to invasive melanoma cells remains elusive. Microphthalmia-associated transcription factor (MITF), is thought to be a central player in melanoma biology, and it controls many aspects of the phenotypic expression of the melanocytic lineage. However, recently the paired box transcription factor PAX3 was shown to transcriptionally activate POU3F2/BRN2, leading to direct repression of MITF expression. Here we present a theory to explain melanoma phenotype switching and discuss the predictions that this theory makes. One prediction is that independent and opposing roles for MITF and PAX3 in melanoma would be expected, and we present empirical evidence supporting this: in melanoma tissues PAX3 expression occurs independently of MITF, and PAX3 does not play a key role in melanoma cell proliferation. Furthermore, we show that knockdown of PAX3 inhibits cell migration in a group of "lower MITF" melanoma cell lines, while knockdown of MITF promotes cell migration in a complementary "higher MITF" group of melanoma cell lines. Moreover, the morphological effects of knocking down PAX3 versus MITF in melanoma cells were found to differ. While these data support the notion of independent roles for MITF and PAX3, additional experiments are required to provide robust examination of the proposed genetic switch theory. Only upon clear delineation of the mechanisms associated with progression and invasion of melanoma cells will successful treatments for invasive melanoma be developed.

  13. Efficient isolation of human metapneumovirus using MNT-1, a human malignant melanoma cell line with early and distinct cytopathic effects.

    PubMed

    Sato, Ko; Watanabe, Oshi; Ohmiya, Suguru; Chiba, Fumiko; Suzuki, Akira; Okamoto, Michiko; Younghuang, Jiang; Hata, Akihiro; Nonaka, Hiroyuki; Kitaoka, Setsuko; Nagai, Yukio; Kawamura, Kazuhisa; Hayashi, Masahiro; Kumaki, Satoru; Suzuki, Tamio; Kawakami, Kazuyoshi; Nishimura, Hidekazu

    2017-11-01

    Isolation of human metapneumovirus (HMPV) from clinical specimens is currently inefficient because of the lack of a cell culture system in which a distinct cytopathic effect (CPE) occurs. The cell lines LLC-MK2, Vero and Vero E6 are used for isolation of HMPV; however, the CPE in these cell lines is subtle and usually requires a long observation period and sometimes blind passages. Thus, a cell line in which an early and distinct CPE occurs following HMPV inoculation is highly desired by clinical virology laboratories. In this study, it was demonstrated that, in the human malignant melanoma cell line MNT-1, obvious syncytium formation occurs shortly after inoculation with HMPV-positive clinical specimens. In addition, the growth and efficiency of isolation of HMPV were greater using MNT-1 than using any other conventional cell line. Addition of this cell line to our routine viral isolation system for clinical specimens markedly enhanced isolation frequency, allowing isolation-based surveillance. MNT-1 has the potential to facilitate clinical and epidemiological studies of HMPV. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    1995-06-15

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

  17. Human Macrophages and Dendritic Cells Can Equally Present MART-1 Antigen to CD8+ T Cells after Phagocytosis of Gamma-Irradiated Melanoma Cells

    PubMed Central

    Barrio, María Marcela; Abes, Riad; Colombo, Marina; Pizzurro, Gabriela; Boix, Charlotte; Roberti, María Paula; Gélizé, Emmanuelle; Rodriguez-Zubieta, Mariana

    2012-01-01

    Dendritic cells (DC) can achieve cross-presentation of naturally-occurring tumor-associated antigens after phagocytosis and processing of dying tumor cells. They have been used in different clinical settings to vaccinate cancer patients. We have previously used gamma-irradiated MART-1 expressing melanoma cells as a source of antigens to vaccinate melanoma patients by injecting irradiated cells with BCG and GM-CSF or to load immature DC and use them as a vaccine. Other clinical trials have used IFN-gamma activated macrophage killer cells (MAK) to treat cancer patients. However, the clinical use of MAK has been based on their direct tumoricidal activity rather than on their ability to act as antigen-presenting cells to stimulate an adaptive antitumor response. Thus, in the present work, we compared the fate of MART-1 after phagocytosis of gamma-irradiated cells by clinical grade DC or MAK as well as the ability of these cells to cross present MART-1 to CD8+ T cells. Using a high affinity antibody against MART-1, 2A9, which specifically stains melanoma tumors, melanoma cell lines and normal melanocytes, the expression level of MART-1 in melanoma cell lines could be related to their ability to stimulate IFN-gamma production by a MART-1 specific HLA-A*0201-restricted CD8+ T cell clone. Confocal microscopy with Alexa Fluor®647-labelled 2A9 also showed that MART-1 could be detected in tumor cells attached and/or fused to phagocytes and even inside these cells as early as 1 h and up to 24 h or 48 h after initiation of co-cultures between gamma-irradiated melanoma cells and MAK or DC, respectively. Interestingly, MART-1 was cross-presented to MART-1 specific T cells by both MAK and DC co-cultured with melanoma gamma-irradiated cells for different time-points. Thus, naturally occurring MART-1 melanoma antigen can be taken-up from dying melanoma cells into DC or MAK and both cell types can induce specific CD8+ T cell cross-presentation thereafter. PMID:22768350

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

    PubMed

    Michelin, Severino; Gallegos, Cristina E; Dubner, Diana; Favier, Benoit; Carosella, Edgardo D

    2009-12-01

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

  19. The Aryl Hydrocarbon Receptor Mediates Leflunomide-Induced Growth Inhibition of Melanoma Cells

    PubMed Central

    O’Donnell, Edmond F.; Kopparapu, Prasad Rao; Koch, Daniel C.; Jang, Hyo Sang; Phillips, Jessica Lynne; Tanguay, Robert L.; Kerkvliet, Nancy I.; Kolluri, Siva Kumar

    2012-01-01

    A novel role of the dihydroorotatedehydrogenase (DHODH) inhibitor leflunomide as a potential anti-melanoma therapy was recently reported (Nature 471∶518-22, 2011). We previously reported that leflunomide strongly activates the transcriptional activity of the Aryl Hydrocarbon Receptor (AhR). We therefore tested whether the AhR regulates the anti-proliferative effects of leflunomide in melanoma. We first evaluated the expression of AhR in melanoma cells and found that AhR is highly expressed in A375 melanoma as well as in several other cancer cell types. To evaluate whether AhR plays a role in regulating the growth inhibitory effects of leflunomide in A375 cells, we generated a stable cell line from parental A375 cells expressing a doxycycline (DOX) inducible AhR shRNA. Using these cells in the absence or presence of DOX (normal AhR levels or AhR-knockdown, respectively) we found that the anti-proliferative effects of leflunomide, but not its metabolite A771726, were strongly dependent upon AhR expression. It has been well established that supplementation of cells with exogenous uridine completely rescues the anti-proliferative effects due to DHODH inhibition. Thus, we performed uridine rescue experiments in A375 cells to determine whether the anti-proliferative effects of leflunomide are solely due to DHODH inhibition as previously reported. Interestingly, saturating levels of uridine only modestly rescued A375 cells from the anti-proliferative effects of both leflunomide and A771726, indicating additional mechanism(s), apart from DHODH inhibition are responsible for the anti-proliferative effects of leflunomide in melanoma cells. Uridine also did not rescue MDA-MB-435S melanoma cell proliferation after leflunomide treatment. Our results reveal that the AhR is a molecular target of leflunomide and support the feasibility of the clinical application of leflunomide for treating melanoma. Furthermore, analysis of expression data from 967 cancer cell lines revealed

  20. Functional Erythropoietin Autocrine Loop in Melanoma

    PubMed Central

    Kumar, Suresh M.; Acs, Geza; Fang, Dong; Herlyn, Meenhard; Elder, David E.; Xu, Xiaowei

    2005-01-01

    Although erythropoietin (Epo) is a known stimulator of erythropoiesis, recent evidence suggests that its biological functions are not confined to hematopoietic cells. To elucidate the role of Epo and erythropoietin receptor (EpoR) in melanoma, we examined the expression and function of these proteins in melanocytes and melanoma cells. We found increased expression of Epo in melanoma cells compared to melanocyte in vitro. EpoR was also strongly expressed in all of the melanoma cell lines and two of the three melanocyte cell lines examined. Epo expression was significantly higher in melanoma than in benign nevi as determined by immunohistochemistry. Although melanoma cells secreted Epo in normoxic condition in vitro, hypoxia and CoCl2 treatment increased Epo secretion. EpoR in melanoma cells was functional, because exogenous Epo increased melanoma resistance to hypoxic stress, pretreatment of melanoma cells with Epo significantly increased resistance to dacarbazine treatment, and Epo increased the phosphorylation of EpoR, RAF, and MEK. In conclusion, we demonstrated constitutive expression of Epo and EpoR as well as autonomous secretion of Epo by melanoma cells, indicating a novel autocrine loop of Epo in melanoma. The results suggest that the autocrine and paracrine functions of Epo might play a role in malignant transformation of melanocytes and in the survival of melanoma cells in hypoxia and other adverse conditions. PMID:15743794

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

    PubMed Central

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

    2007-01-01

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

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

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

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

    2013-07-01

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

  3. ZEB1-mediated melanoma cell plasticity enhances resistance to MAPK inhibitors.

    PubMed

    Richard, Geoffrey; Dalle, Stéphane; Monet, Marie-Ambre; Ligier, Maud; Boespflug, Amélie; Pommier, Roxane M; de la Fouchardière, Arnaud; Perier-Muzet, Marie; Depaepe, Lauriane; Barnault, Romain; Tondeur, Garance; Ansieau, Stéphane; Thomas, Emilie; Bertolotto, Corine; Ballotti, Robert; Mourah, Samia; Battistella, Maxime; Lebbé, Céleste; Thomas, Luc; Puisieux, Alain; Caramel, Julie

    2016-10-01

    Targeted therapies with MAPK inhibitors (MAPKi) are faced with severe problems of resistance in BRAF-mutant melanoma. In parallel to the acquisition of genetic mutations, melanoma cells may also adapt to the drugs through phenotype switching. The ZEB1 transcription factor, a known inducer of EMT and invasiveness, is now considered as a genuine oncogenic factor required for tumor initiation, cancer cell plasticity, and drug resistance in carcinomas. Here, we show that high levels of ZEB1 expression are associated with inherent resistance to MAPKi in BRAF V 600 -mutated cell lines and tumors. ZEB1 levels are also elevated in melanoma cells with acquired resistance and in biopsies from patients relapsing while under treatment. ZEB1 overexpression is sufficient to drive the emergence of resistance to MAPKi by promoting a reversible transition toward a MITF low /p75 high stem-like and tumorigenic phenotype. ZEB1 inhibition promotes cell differentiation, prevents tumorigenic growth in vivo, sensitizes naive melanoma cells to MAPKi, and induces cell death in resistant cells. Overall, our results demonstrate that ZEB1 is a major driver of melanoma cell plasticity, driving drug adaptation and phenotypic resistance to MAPKi. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

  4. Combined treatment with Ad-hTRAIL and DTIC or SAHA is associated with increased mitochondrial-mediated apoptosis in human melanoma cell lines.

    PubMed

    Lillehammer, Trine; Engesaeter, Birgit O; Prasmickaite, Lina; Maelandsmo, Gunhild M; Fodstad, Oystein; Engebraaten, Olav

    2007-06-01

    Currently, dacarbazine (DTIC) is the only approved systemic treatment for metastatic malignant melanoma. However, the modest treatment effect encourages studies on novel therapeutic molecules, delivery systems and combination therapies. Full-length TRAIL, delivered from an adenoviral vector (Ad-hTRAIL), was studied in combination with DTIC or the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) in human melanoma cell lines. The cytotoxic potential of the combination treatments was assessed by cell viability measurements and CalcuSyn analysis. Involvement of apoptosis was analyzed by TUNEL staining, mitochondrial membrane potential measurements, and activation and expression levels of caspases and other mediators of apoptosis. Ad-hTRAIL in combination with DTIC or SAHA resulted in additive or synergistic growth inhibition compared to each treatment used as single agent. Both combinations augmented apoptosis, which was mediated through the death receptor (DR) pathway by enhanced activation of caspase-8, and through increased loss of mitochondrial integrity. Provoked cleavage of Bid, which bridges the extrinsic and intrinsic apoptosis pathways, and downregulation of the anti-apoptotic mediators Bcl-X(L), Mcl-1 and XIAP (but not Bcl-2) were critical contributing factors. Increased levels of DR4 and DR5 were not a common underlying mechanism as DTIC did not affect the levels of either of the receptors. However, SAHA-induced expression of DR4 may have reduced the TRAIL resistance in the SKMEL-28 cell line. Administration of Ad-hTRAIL in combination with DTIC or SAHA enhances apoptosis in human melanoma cell lines, and suggests that the therapeutic potential of such treatment strategies should be further evaluated for possible clinical use.

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2012-01-01

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

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

    PubMed

    Risberg, Karianne; Fodstad, Oystein; Andersson, Yvonne

    2010-04-01

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

  8. Anacardic acid enhances the anticancer activity of liposomal mitoxantrone towards melanoma cell lines – in vitro studies

    PubMed Central

    Legut, Mateusz; Lipka, Dominik; Filipczak, Nina; Piwoni, Adriana; Kozubek, Arkadiusz; Gubernator, Jerzy

    2014-01-01

    This paper describes a novel formulation of antineoplastic drug: mitoxantrone loaded into liposomal carriers enriched with encapsulated anacardic acid in the liposomal bilayer using a vitamin C gradient. Anacardic acid is a potent epigenetic agent with anticancer activity. This is the first liposomal formulation to combine an actively encapsulated drug and anacardic acid. The liposomes were characterized in terms of basic parameters, such as size, zeta potential, optimal drug-to-lipid ratio, loading time and temperature, and stability at 4°C and in human plasma in vitro. The formulation was found to be stable, and the loading process was rapid and efficient (drug-to-lipid ratio of up to 0.3 with over 90% efficiency in 5 minutes). The cytotoxicity of these formulations was assessed using the human melanoma cell lines A375 and Hs294T and the normal human dermal fibroblast line. The results showed that anacardic acid and to a smaller extent vitamin C significantly increased the cytotoxicity of the drug towards melanoma compared to ammonium sulfate liposomes. On the other hand, vitamin C and anacardic acid both protected normal cells from damage caused by the drug. The formulation combining anacardic acid, vitamin C, and mitoxantrone showed promising results in terms of cytotoxicity and cytoprotection. Therefore, it has potential for anticancer treatment. PMID:24489469

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

    PubMed

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

    2012-02-15

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

  10. Fibroblasts Protect Melanoma Cells from the Cytotoxic Effects of Doxorubicin

    PubMed Central

    Tiago, Manoela; de Oliveira, Edson Mendes; Brohem, Carla Abdo; Pennacchi, Paula Comune; Paes, Rafael Duarte; Haga, Raquel Brandão; Campa, Ana; de Moraes Barros, Silvia Berlanga; Smalley, Keiran S.

    2014-01-01

    Melanoma is the most aggressive form of skin cancer and until recently, it was extremely resistant to radio-, immuno-, and chemotherapy. Despite the latest success of BRAF V600E-targeted therapies, responses are typically short lived and relapse is all but certain. Furthermore, a percentage (40%) of melanoma cells is BRAF wild type. Emerging evidence suggests a role for normal host cells in the occurrence of drug resistance. In the current study, we compared a variety of cell culture models with an organotypic incomplete skin culture model (the “dermal equivalent”) to investigate the role of the tissue microenvironment in the response of melanoma cells to the chemotherapeutic agent doxorubicin (Dox). In the dermal equivalent model, consisting of fibroblasts embedded in type I collagen matrix, melanoma cells showed a decreased cytotoxic response when compared with less complex culture conditions, such as seeding on plastic cell culture plate (as monolayers cultures) or on collagen gel. We further investigated the role of the microenvironment in p53 induction and caspase 3 and 9 cleavage. Melanoma cell lines cultured on dermal equivalent showed decreased expression of p53 after Dox treatment, and this outcome was accompanied by induction of interleukin IL-6, IL-8, and matrix metalloproteinases 2 and 9. Here, we show that the growth of melanoma cells in the dermal equivalent model inflects drug responses by recapitulating important pro-survival features of the tumor microenvironment. These studies indicate that the presence of stroma enhances the drug resistance of melanoma in vitro, more closely mirroring the in vivo phenotype. Our data, thus, demonstrate the utility of organotypic cell culture models in providing essential context-dependent information critical for the development of new therapeutic strategies for melanoma. We believe that the organotypic model represents an improved screening platform to investigate novel anti-cancer agents, as it provides

  11. Differential response of human melanoma and Ehrlich ascites cells in vitro to the ribosome-inactivating protein luffin.

    PubMed

    Poma, A; Miranda, M; Spanò, L

    1998-10-01

    The cytotoxicity and inhibitory effect on proliferation of the type 1 ribosome-inactivating protein luffin purified from the seeds of Luffa aegyptiaca were investigated both in human metastatic melanoma cells and in murine Ehrlich ascites tumour cells. Results indicate that luffin from the seeds of Luffa aegyptiaca is cytotoxic to the cell lines tested, with approximately 10 times greater potency in Ehrlich cells. Luffin was found to induce an increase in cytosolic oligonucleosome-bound DNA in both melanoma and Ehrlich ascites tumour cells, the level of DNA fragmentation in the former cell line being higher than in the latter. Experiments with melanoma cells indicate that an increase in cytosolic nucleosomes could be supportive of apoptosis as the type of cell death induced by luffin.

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

    PubMed

    Taghizadeh, Rouzbeh; Noh, Minsoo; Huh, Yang Hoon; Ciusani, Emilio; Sigalotti, Luca; Maio, Michele; Arosio, Beatrice; Nicotra, Maria R; Natali, PierGiorgio; Sherley, James L; La Porta, Caterina A M

    2010-12-22

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

  13. Silymarin Targets β-Catenin Signaling in Blocking Migration/Invasion of Human Melanoma Cells

    PubMed Central

    Vaid, Mudit; Prasad, Ram; Sun, Qian; Katiyar, Santosh K.

    2011-01-01

    Metastatic melanoma is a leading cause of death from skin diseases, and is often associated with activation of Wnt/β-catenin signaling pathway. We have examined the inhibitory effect of silymarin, a plant flavanoid from Silybum marianum, on cell migration of metastasis-specific human melanoma cell lines (A375 and Hs294t) and assessed whether Wnt/β-catenin signaling is the target of silymarin. Using an in vitro invasion assay, we found that treatment of human melanoma cell lines with silymarin resulted in concentration-dependent inhibition of cell migration, which was associated with accumulation of cytosolic β-catenin, while reducing the nuclear accumulation of β-catenin (i.e., β-catenin inactivation) and reducing the levels of matrix metalloproteinase (MMP) -2 and MMP-9 which are the down-stream targets of β-catenin. Silymarin enhanced: (i) the levels of casein kinase 1α, glycogen synthase kinase-3β and phosphorylated-β-catenin on critical residues Ser45, Ser33/37 and Thr41, and (ii) the binding of β-transducin repeat-containing proteins (β-TrCP) with phospho forms of β-catenin in melanoma cells. These events play important roles in degradation or inactivation of β-catenin. To verify whether β-catenin is a potent molecular target of silymarin, the effect of silymarin was determined on β-catenin-activated (Mel 1241) and β-catenin-inactivated (Mel 1011) melanoma cells. Treatment of Mel 1241 cells with silymarin or FH535, an inhibitor of Wnt/β-catenin pathway, significantly inhibited cell migration of Mel 1241 cells, which was associated with the elevated levels of casein kinase 1α and glycogen synthase kinase-3β, and decreased accumulation of nuclear β-catenin and inhibition of MMP-2 and MMP-9 levels. However, this effect of silymarin and FH535 was not found in Mel 1011 melanoma cells. These results indicate for the first time that silymarin inhibits melanoma cell migration by targeting β-catenin signaling pathway. PMID:21829575

  14. Cytotoxic effect of the serotonergic drug 1-(1-Naphthyl)piperazine against melanoma cells.

    PubMed

    Menezes, Ana Catarina; Carvalheiro, Manuela; Ferreira de Oliveira, José Miguel P; Ascenso, Andreia; Oliveira, Helena

    2018-03-01

    1-(1-Naphthyl)piperazine (1-NPZ) is a serotonergic derivative of quipazine acting both as antagonist and agonist of different serotonin receptors, with promising results for the management of skin cancer. In this work, we studied the effect of 1-NPZ on human MNT-1 melanoma cells by evaluating its effects on cell viability, ability to form colonies, cell cycle dynamics, reactive oxygen species (ROS) production and apoptosis. Treatment of MNT-1 cells with 1-NPZ for 24h decreased cell viability and induced apoptosis in a dose-dependent manner. Activity against melanoma was confirmed with a different melanoma cell line, SK-MEL-28. Simultaneously, 1-NPZ affected cell cycle progression by mediating a S-phase delay. Higher levels of ROS were also detected in MNT-1 cells after treatment with 1-NPZ. Furthermore, 1-NPZ significantly increased the expression of cyclooxygenase-2 in MNT-1 cells. These findings suggest that 1-NPZ pretreatment is able to induce oxidative stress, and consequently apoptotic cell death in melanoma cells. In conclusion, this study demonstrates the cytotoxic and genotoxic potential of 1-NPZ against melanoma cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Genetically fluorescent melanoma bone and organ metastasis models.

    PubMed

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

    1999-11-01

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

  16. Systems analysis of apoptosis protein expression allows the case-specific prediction of cell death responsiveness of melanoma cells

    PubMed Central

    Passante, E; Würstle, M L; Hellwig, C T; Leverkus, M; Rehm, M

    2013-01-01

    Many cancer entities and their associated cell line models are highly heterogeneous in their responsiveness to apoptosis inducers and, despite a detailed understanding of the underlying signaling networks, cell death susceptibility currently cannot be predicted reliably from protein expression profiles. Here, we demonstrate that an integration of quantitative apoptosis protein expression data with pathway knowledge can predict the cell death responsiveness of melanoma cell lines. By a total of 612 measurements, we determined the absolute expression (nM) of 17 core apoptosis regulators in a panel of 11 melanoma cell lines, and enriched these data with systems-level information on apoptosis pathway topology. By applying multivariate statistical analysis and multi-dimensional pattern recognition algorithms, the responsiveness of individual cell lines to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or dacarbazine (DTIC) could be predicted with very high accuracy (91 and 82% correct predictions), and the most effective treatment option for individual cell lines could be pre-determined in silico. In contrast, cell death responsiveness was poorly predicted when not taking knowledge on protein–protein interactions into account (55 and 36% correct predictions). We also generated mathematical predictions on whether anti-apoptotic Bcl-2 family members or x-linked inhibitor of apoptosis protein (XIAP) can be targeted to enhance TRAIL responsiveness in individual cell lines. Subsequent experiments, making use of pharmacological Bcl-2/Bcl-xL inhibition or siRNA-based XIAP depletion, confirmed the accuracy of these predictions. We therefore demonstrate that cell death responsiveness to TRAIL or DTIC can be predicted reliably in a large number of melanoma cell lines when investigating expression patterns of apoptosis regulators in the context of their network-level interplay. The capacity to predict responsiveness at the cellular level may contribute to

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

    PubMed Central

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

    2014-01-01

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

  18. In vitro effects of histone deacetylase inhibitors and mitomycin C on tenon capsule fibroblasts and conjunctival melanoma cells.

    PubMed

    Cunneen, Thomas S; Conway, R Max; Madigan, Michele C

    2009-04-01

    To investigate the effects of mitomycin C and the histone deacetylase inhibitors sodium butyrate and trichostatin on the viability and growth of conjunctival melanoma cell lines and Tenon capsule fibroblasts. Cells were treated with a range of concentrations of sodium butyrate, trichostatin, and mitomycin C. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) assays were performed 48 hours after treatment. Treated cells were stained with acridine orange/ethidium bromide to assess for cell death. Cell-cycle changes in histone deacetylase inhibitor-treated melanoma cells were quantified using flow cytometry. All agents induced dose-dependent cell death in the melanoma cell lines; however, sodium butyrate and trichostatin were relatively nontoxic to Tenon capsule fibroblasts. Acridine orange/ethidium bromide staining indicated that sodium butyrate and trichostatin induced apoptotic cell death. At low doses, sodium butyrate and trichostatin induced a G1 cell-cycle block in the melanoma cells. Sodium butyrate and trichostatin induced cell death in melanoma cells, comparable with mitomycin C, with minimal effect on Tenon capsule fibroblasts. In addition, they induced a G1 cell-cycle block. These findings support the need for further investigation into the in vivo efficacy of these agents.

  19. The Broad Spectrum Receptor Tyrosine Kinase Inhibitor Dovitinib Suppresses Growth of BRAF Mutant Melanoma Cells in Combination with Other Signaling Pathway Inhibitors

    PubMed Central

    Langdon, Casey G.; Held, Matthew A.; Platt, James T.; Meeth, Katrina; Iyidogan, Pinar; Mamillapalli, Ramanaiah; Koo, Andrew B.; Klein, Michael; Liu, Zongzhi; Bosenberg, Marcus W.; Stern, David F.

    2016-01-01

    Summary BRAF inhibitors have revolutionized treatment of mutant BRAF metastatic melanomas. However, resistance develops rapidly following BRAF inhibitor treatment. We have found that BRAF-mutant melanoma cell lines are more sensitive than wild-type BRAF cells to the small molecule tyrosine kinase inhibitor dovitinib. Sensitivity is associated with inhibition of a series of known dovitinib targets. Dovitinib in combination with several agents inhibits growth more effectively than either agent alone. These combinations inhibit BRAF-mutant melanoma and colorectal carcinoma cell lines, including cell lines with intrinsic or selected BRAF inhibitor resistance. Hence, combinations of dovitinib with second agents are potentially effective therapies for BRAF-mutant melanomas, regardless of their sensitivity to BRAF inhibitors. PMID:25854919

  20. The fragile X mental retardation protein regulates tumor invasiveness-related pathways in melanoma cells.

    PubMed

    Zalfa, Francesca; Panasiti, Vincenzo; Carotti, Simone; Zingariello, Maria; Perrone, Giuseppe; Sancillo, Laura; Pacini, Laura; Luciani, Flavie; Roberti, Vincenzo; D'Amico, Silvia; Coppola, Rosa; Abate, Simona Osella; Rana, Rosa Alba; De Luca, Anastasia; Fiers, Mark; Melocchi, Valentina; Bianchi, Fabrizio; Farace, Maria Giulia; Achsel, Tilmann; Marine, Jean-Christophe; Morini, Sergio; Bagni, Claudia

    2017-11-16

    The fragile X mental retardation protein (FMRP) is lacking or mutated in patients with the fragile X syndrome (FXS), the most frequent form of inherited intellectual disability. FMRP affects metastasis formation in a mouse model for breast cancer. Here we show that FMRP is overexpressed in human melanoma with high Breslow thickness and high Clark level. Furthermore, meta-analysis of the TCGA melanoma data revealed that high levels of FMRP expression correlate significantly with metastatic tumor tissues, risk of relapsing and disease-free survival. Reduction of FMRP in metastatic melanoma cell lines impinges on cell migration, invasion and adhesion. Next-generation sequencing in human melanoma cells revealed that FMRP regulates a large number of mRNAs involved in relevant processes of melanoma progression. Our findings suggest an association between FMRP levels and the invasive phenotype in melanoma and might open new avenues towards the discovery of novel therapeutic targets.

  1. The fragile X mental retardation protein regulates tumor invasiveness-related pathways in melanoma cells

    PubMed Central

    Zalfa, Francesca; Panasiti, Vincenzo; Carotti, Simone; Zingariello, Maria; Perrone, Giuseppe; Sancillo, Laura; Pacini, Laura; Luciani, Flavie; Roberti, Vincenzo; D'Amico, Silvia; Coppola, Rosa; Abate, Simona Osella; Rana, Rosa Alba; De Luca, Anastasia; Fiers, Mark; Melocchi, Valentina; Bianchi, Fabrizio; Farace, Maria Giulia; Achsel, Tilmann; Marine, Jean-Christophe; Morini, Sergio; Bagni, Claudia

    2017-01-01

    The fragile X mental retardation protein (FMRP) is lacking or mutated in patients with the fragile X syndrome (FXS), the most frequent form of inherited intellectual disability. FMRP affects metastasis formation in a mouse model for breast cancer. Here we show that FMRP is overexpressed in human melanoma with high Breslow thickness and high Clark level. Furthermore, meta-analysis of the TCGA melanoma data revealed that high levels of FMRP expression correlate significantly with metastatic tumor tissues, risk of relapsing and disease-free survival. Reduction of FMRP in metastatic melanoma cell lines impinges on cell migration, invasion and adhesion. Next-generation sequencing in human melanoma cells revealed that FMRP regulates a large number of mRNAs involved in relevant processes of melanoma progression. Our findings suggest an association between FMRP levels and the invasive phenotype in melanoma and might open new avenues towards the discovery of novel therapeutic targets. PMID:29144507

  2. Expression of the chondroitin sulphate proteoglycan molecular complex in six human melanoma xenograft lines studied by flow cytometry and immunohistochemistry.

    PubMed

    Nagelhus, T A; Rofstad, E K

    1993-06-01

    The expression of the chondroitin sulphate proteoglycan (CSP) molecular complex in six human melanoma xenograft lines (BEX-t, COX-t, HUX-t, ROX-t, SAX-t, WIX-t) was studied by flow cytometry and immunohistochemistry using the monoclonal antibodies 9.2.27, ME31.3, G7A5, and NKI.M6. The two methods and the four antibodies gave consistent results. The six melanoma lines could be divided into three distinct groups of two lines each; expression was high in the HUX-t and ROX-t lines and intermediate in the BEX-t and SAX-t lines, whereas the COX-t and WIX-t lines were negative. The mean number of epitopes per cell for 9.2.27 was approximately twice as high as for ME31.3, G7A5, and NKI.M6 and was estimated to range from 0.8 +/- 0.1 x 10(5) to 1.9 +/- 0.2 x 10(5) in the positive xenograft lines. The expression of the CSP complex was heterogeneous. The immunofluorescence histograms measured by flow cytometry were therefore broad for all tumour lines. A significant fraction of the HUX-t cells was negative or weakly stained. These cells appeared as clear negative patches in the immunohistochemical preparations. Moreover, most morphologically intact tumour cells adjacent to necrotic areas did not show significant expression of the CSP complex, irrespective of tumour line. These cells were probably hypoxic and thus resistant to radiation therapy. The expression of the CSP complex in the xenograft lines was similar to that reported for melanoma in man.

  3. Defective Cell Cycle Checkpoint Functions in Melanoma Are Associated with Altered Patterns of Gene Expression

    PubMed Central

    Kaufmann, William K.; Nevis, Kathleen R.; Qu, Pingping; Ibrahim, Joseph G.; Zhou, Tong; Zhou, Yingchun; Simpson, Dennis A.; Helms-Deaton, Jennifer; Cordeiro-Stone, Marila; Moore, Dominic T.; Thomas, Nancy E.; Hao, Honglin; Liu, Zhi; Shields, Janiel M.; Scott, Glynis A.; Sharpless, Norman E.

    2009-01-01

    Defects in DNA damage responses may underlie genetic instability and malignant progression in melanoma. Cultures of normal human melanocytes (NHMs) and melanoma lines were analyzed to determine whether global patterns of gene expression could predict the efficacy of DNA damage cell cycle checkpoints that arrest growth and suppress genetic instability. NHMs displayed effective G1 and G2 checkpoint responses to ionizing radiation-induced DNA damage. A majority of melanoma cell lines (11/16) displayed significant quantitative defects in one or both checkpoints. Melanomas with B-RAF mutations as a class displayed a significant defect in DNA damage G2 checkpoint function. In contrast the epithelial-like subtype of melanomas with wild-type N-RAS and B-RAF alleles displayed an effective G2 checkpoint but a significant defect in G1 checkpoint function. RNA expression profiling revealed that melanoma lines with defects in the DNA damage G1 checkpoint displayed reduced expression of p53 transcriptional targets, such as CDKN1A and DDB2, and enhanced expression of proliferation-associated genes, such as CDC7 and GEMININ. A Bayesian analysis tool was more accurate than significance analysis of microarrays for predicting checkpoint function using a leave-one-out method. The results suggest that defects in DNA damage checkpoints may be recognized in melanomas through analysis of gene expression. PMID:17597816

  4. Tumor Cell Plasticity in Uveal Melanoma

    PubMed Central

    Folberg, Robert; Arbieva, Zarema; Moses, Jonas; Hayee, Amin; Sandal, Tone; Kadkol, ShriHari; Lin, Amy Y.; Valyi-Nagy, Klara; Setty, Suman; Leach, Lu; Chévez-Barrios, Patricia; Larsen, Peter; Majumdar, Dibyen; Pe’er, Jacob; Maniotis, Andrew J.

    2006-01-01

    The histological detection of laminin-rich vasculogenic mimicry patterns in human primary uveal melanomas is associated with death from metastases. We therefore hypothesized that highly invasive uveal melanoma cells forming vasculogenic mimicry patterns after exposure to a laminin-rich three-dimensional microenvironment would differentially express genes associated with invasive and metastatic behavior. However, we discovered that genes associated with differentiation (GDF15 and ATF3) and suppression of proliferation (CDKNa1/p21) were up-regulated in highly invasive uveal melanoma cells forming vasculogenic mimicry patterns, and genes associated with promotion of invasive and metastatic behavior such as CD44, CCNE2 (cyclin E2), THBS1 (thrombospondin 1), and CSPG2 (chondroitin sulfate proteoglycan; versican) were down-regulated. After forming vasculogenic mimicry patterns, uveal melanoma cells invaded only short distances, failed to replicate, and changed morphologically from the invasive epithelioid to the indolent spindle A phenotype. In human tissue samples, uveal melanoma cells within vasculogenic mimicry patterns assumed the spindle A morphology, and the expression of Ki67 was significantly reduced in adjacent melanoma cells. Thus, the generation of vasculogenic mimicry patterns is accompanied by dampening of the invasive and metastatic uveal melanoma genotype and phenotype and underscores the plasticity of these cells in response to cues from the microenvironment. PMID:17003493

  5. CD63 tetraspanin is a negative driver of epithelial-to-mesenchymal transition in human melanoma cells.

    PubMed

    Lupia, Antonella; Peppicelli, Silvia; Witort, Ewa; Bianchini, Francesca; Carloni, Vinicio; Pimpinelli, Nicola; Urso, Carmelo; Borgognoni, Lorenzo; Capaccioli, Sergio; Calorini, Lido; Lulli, Matteo

    2014-12-01

    The CD63 tetraspanin is highly expressed in the early stages of melanoma and decreases in advanced lesions, suggesting it as a possible suppressor of tumor progression. We employed loss- and gain-of-gene-function approaches to investigate the role of CD63 in melanoma progression and acquisition of the epithelial-to-mesenchymal transition (EMT) program. We used two human melanoma cell lines derived from primary tumors and one primary human melanoma cell line isolated from a cutaneous metastasis, differing by levels of CD63 expression. CD63-silenced melanoma cells showed enhanced motility and invasiveness with downregulation of E-cadherin and upregulation of N-cadherin and Snail. In parallel experiments, transient and stable ectopic expression of CD63 resulted in a robust reduction of cell motility, invasiveness, and protease activities, which was proportional to the increase in CD63 protein level. Transfected cells overexpressing the highest level of CD63 when transplanted into immunodeficient mice showed a reduced incidence and rate of tumor growth. Moreover, these cells showed a reduction of N-cadherin, Vimentin, Zeb1, and a-SMA, and a significant resistance to undergo an EMT program both in basal condition and in the following stimulation with TGFβ. Thus, our results establish a previously unreported mechanistic link between the tetraspanin CD63 and EMT abrogation in melanoma.

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

    PubMed

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

    2016-06-01

    A majority of patients with melanoma brain metastases develop multiple lesions, and these patients show particularly poor prognosis. To develop improved treatment strategies, detailed insights into the biology of melanoma brain metastases, and particularly the development of multiple lesions, are needed. The purpose of this preclinical investigation was to study melanoma cell migration within the brain after cell injection into a well-defined intracerebral site. A-07, D-12, R-18, and U-25 human melanoma cells transfected with green fluorescent protein were injected stereotactically into the right cerebral hemisphere of nude mice. Moribund mice were killed and autopsied, and the brain was evaluated by fluorescence imaging or histological examination. Intracerebral inoculation of melanoma cells produced multiple lesions involving all regions of the brain, suggesting that the cells were able to migrate over substantial distances within the brain. Multiple modes of transport were identified, and all transport modes were observed in all four melanoma lines. Thus, the melanoma cells were passively transported via the flow of cerebrospinal fluid in the meninges and ventricles, they migrated actively along leptomeningeal and brain parenchymal blood vessels, and they migrated actively along the surfaces separating different brain compartments. Migration of melanoma cells after initial arrest, extravasation, and growth at a single location within the brain may contribute significantly to the development of multiple melanoma brain metastases. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  7. Mitochondrial oxidative stress is the achille's heel of melanoma cells resistant to Braf-mutant inhibitor

    PubMed Central

    André, Fanny; Jonneaux, Aurélie; Scalbert, Camille; Garçon, Guillaume; Malet-Martino, Myriam; Balayssac, Stéphane; Rocchi, Stephane; Savina, Ariel; Formstecher, Pierre; Mortier, Laurent; Kluza, Jérome; Marchetti, Philippe

    2013-01-01

    Vemurafenib/PLX4032, a selective inhibitor of mutant BRAFV600E, constitutes a paradigm shift in melanoma therapy. Unfortunately, acquired resistance, which unavoidably occurs, represents one major limitation to clinical responses. Recent studies have highlighted that vemurafenib activated oxidative metabolism in BRAFV600E melanomas expressing PGC1α. However, the oxidative state of melanoma resistant to BRAF inhibitors is unknown. We established representative in vitro and in vivo models of human melanoma resistant to vemurafenib including primary specimens derived from melanoma patients. Firstly, our study reveals that vemurafenib increased mitochondrial respiration and ROS production in BRAFV600E melanoma cell lines regardless the expression of PGC1α. Secondly, melanoma cells that have acquired resistance to vemurafenib displayed intrinsically high rates of mitochondrial respiration associated with elevated mitochondrial oxidative stress irrespective of the presence of vemurafenib. Thirdly, the elevated ROS level rendered vemurafenib-resistant melanoma cells prone to cell death induced by pro-oxidants including the clinical trial drug, elesclomol. Based on these observations, we propose that the mitochondrial oxidative signature of resistant melanoma constitutes a novel opportunity to overcome resistance to BRAF inhibition. PMID:24161908

  8. Hypoxia increases the heterogeneity of melanoma cell populations and affects the response to vemurafenib.

    PubMed

    Pucciarelli, Daniela; Lengger, Nina; Takáčová, Martina; Csaderova, Lucia; Bartosova, Maria; Breiteneder, Heimo; Pastorekova, Silvia; Hafner, Christine

    2016-04-01

    A hypoxic microenvironment is one of the predominant reasons for incomplete response to melanoma treatment. Vemurafenib, which targets the mutated BRAF-V600 kinase, improves melanoma patient survival, however, resistance invariably develops. The present study evaluated the effect of hypoxia on three BRAF-V600E mutant melanoma cell lines, M14, A375 and 518A2, treated with vemurafenib. Compared with the other two cell lines, hypoxic vemurafenib-treated A375 cells exhibited an enhanced cell proliferation rate and migratory capacity compared with normoxic vemurafenib-treated A375 cells. Immunoblotting analyses revealed that the expression levels of hypoxia inducible factor (HIF)1α and carbonic anhydrase IX were reduced in vemurafenib‑treated M14 and 518A2 cells, however, not in A375 cells. The expression levels of the mitogen‑activated protein kinase, Janus kinase-signal transducer and activator of transcription, and phosphatidylinositol-4,5-bisphosphate 3‑kinase signaling pathway proteins revealed a cell‑type specific response to vemurafenib and hypoxia. Knockdown experiments of HIF1α performed in hypoxic A375 cells decreased the expression of phosphorylated (p‑)protein kinase B, which was restored following vemurafenib treatment, and increased the expression of p‑extracellular‑signal‑regulated kinases. Therefore, three melanoma cell lines responded to vemurafenib under hypoxia in a cell type‑specific manner, suggesting that a subset of cells provides a treatment-resistant pool, from which disease relapse may originate. These data confirmed that vemurafenib may be successful in treating the proliferating cells, whereas the non‑proliferating subpopulation must be addressed by a combination of vemurafenib with other treatment strategies.

  9. A natural product-like JAK2/STAT3 inhibitor induces apoptosis of malignant melanoma cells

    PubMed Central

    Zhong, Hai-Jing; Dong, Zhen-Zhen; Vellaisamy, Kasipandi; Lu, Jin-Jian; Chen, Xiu-Ping; Chiu, Pauline; Kwong, Daniel W. J.; Han, Quan-Bin; Ma, Dik-Lung

    2017-01-01

    The JAK2/STAT3 signaling pathway plays a critical role in tumorigenesis, and has been suggested as a potential molecular target for anti-melanoma therapeutics. However, few JAK2 inhibitors were being tested for melanoma therapy. In this study, eight amentoflavone analogues were evaluated for their activity against human malignant melanoma cells. The most potent analogue, compound 1, inhibited the phosphorylation of JAK2 and STAT3 in human melanoma cells, but had no discernible effect on total JAK2 and STAT3 levels. A cellular thermal shift assay was performed to identify that JAK2 is engaged by 1 in cell lysates. Moreover, compound 1 showed higher antiproliferative activity against human melanoma A375 cells compared to a panel of cancer and normal cell lines. Compound 1 also activated caspase-3 and cleaved PARP, which are markers of apoptosis, and suppressed the anti-apoptotic Bcl-2 level. Finally, compound 1 induced apoptosis in 80% of treated melanoma cells. To our knowledge, compound 1 is the first amentoflavone-based JAK2 inhibitor to be investigated for use as an anti-melanoma agent. PMID:28570563

  10. Antiproliferative Activity of Cyanophora paradoxa Pigments in Melanoma, Breast and Lung Cancer Cells

    PubMed Central

    Baudelet, Paul-Hubert; Gagez, Anne-Laure; Bérard, Jean-Baptiste; Juin, Camille; Bridiau, Nicolas; Kaas, Raymond; Thiéry, Valérie; Cadoret, Jean-Paul; Picot, Laurent

    2013-01-01

    The glaucophyte Cyanophora paradoxa (Cp) was chemically investigated to identify pigments efficiently inhibiting malignant melanoma, mammary carcinoma and lung adenocarcinoma cells growth. Cp water and ethanol extracts significantly inhibited the growth of the three cancer cell lines in vitro, at 100 µg·mL−1. Flash chromatography of the Cp ethanol extract, devoid of c-phycocyanin and allophycocyanin, enabled the collection of eight fractions, four of which strongly inhibited cancer cells growth at 100 µg·mL−1. Particularly, two fractions inhibited more than 90% of the melanoma cells growth, one inducing apoptosis in the three cancer cells lines. The detailed analysis of Cp pigment composition resulted in the discrimination of 17 molecules, ten of which were unequivocally identified by high resolution mass spectrometry. Pheophorbide a, β-cryptoxanthin and zeaxanthin were the three main pigments or derivatives responsible for the strong cytotoxicity of Cp fractions in cancer cells. These data point to Cyanophora paradoxa as a new microalgal source to purify potent anticancer pigments, and demonstrate for the first time the strong antiproliferative activity of zeaxanthin and β-cryptoxanthin in melanoma cells. PMID:24189278

  11. RASSF6 exhibits promoter hypermethylation in metastatic melanoma and inhibits invasion in melanoma cells

    PubMed Central

    Mezzanotte, Jessica J; Hill, Victoria; Schmidt, M Lee; Shinawi, Thoraia; Tommasi, Stella; Krex, Dietmar; Schackert, Gabriele; Pfeifer, Gerd P; Latif, Farida; Clark, Geoffrey J

    2014-01-01

    Brain metastasis is a major contributor to cancer mortality, yet, the genetic changes underlying the development of this capacity remain poorly understood. RASSF proteins are a family of tumor suppressors that often suffer epigenetic inactivation during tumorigenesis. However, their epigenetic status in brain metastases has not been well characterized. We have examined the promoter methylation of the classical RASSF members (RASSF1A-RASSF6) in a panel of metastatic brain tumor samples. RASSF1A and RASSF2 have been shown to undergo promoter methylation at high frequency in primary lung and breast tumors and in brain metastases. Other members exhibited little or no methylation in these tumors. In examining melanoma metastases, however, we found that RASSF6 exhibits the highest frequency of inactivation in melanoma and in melanoma brain metastases. Most melanomas are driven by an activating mutation in B-Raf. Introduction of RASSF6 into a B-RafV600E-containing metastatic melanoma cell line inhibited its ability to invade through collagen and suppressed MAPK pathway activation and AKT. RASSF6 also appears to increase the association of mutant B-Raf and MST1, providing a potential mechanism by which RASSF6 is able to suppress MAPK activation. Thus, we have identified a novel potential role for RASSF6 in melanoma development. Promoter methylation leading to reduced expression of RASSF6 may play an important role in melanoma development and may contribute to brain metastases. PMID:25482183

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Kanoh, Maho; Amoh, Yasuyuki; Tanabe, Kenichi; Maejima, Hideki; Takasu, Hiroshi; Katsuoka, Kensei

    2010-06-01

    Nestin, a marker of neural stem cells, is expressed in the stem cells of the mouse hair follicle. The nestin-expressing hair follicle stem cells can differentiate into neurons, glia, keratocytes, smooth muscle cells and melanocytes in vitro. These pluripotent nestin-expressing stem cells are keratin 15 (K15)-negative, suggesting that they are in a relatively undifferentiated state. Recent studies suggest that the epithelial stem cells are important in tumorigenesis, and nestin expression is thought to be important in tumorigenesis. In the present study, we examined the expression of the hair follicle and neural stem cell marker nestin, as well as S-100 and HMB-45, in melanoma. Nestin immunoreactivity was observed in the HMB-45-negative melanoma cells in all five cases of amelanotic nodular melanomas. Moreover, nestin immunoreactivity was observed in the dermal parts in seven of 10 cases of melanotic nodular melanomas. Especially, nestin immunoreactivity was observed in the HMB-45-negative melanoma cells in the dermal parts of all 10 cases of HMB-45-negative amelanotic and melanotic nodular melanomas. On the other hand, nestin expression was negative in 10 of 12 cases of superficial spreading melanoma. These results suggest that nestin is an important marker of HMB-45-negative melanoma cells in the dermal parts of patients with nodular melanoma.

  14. Resveratrol Overcomes Cellular Resistance to Vemurafenib Through Dephosphorylation of AKT in BRAF-mutated Melanoma Cells.

    PubMed

    Luo, Hao; Umebayashi, Masayo; Doi, Keiko; Morisaki, Takashi; Shirasawa, Senji; Tsunoda, Toshiyuki

    2016-07-01

    The serine/threonine-protein kinase B-Raf (BRAF) V600E mutant (BRAF(V600E)) inhibitor vemurafenib, has improved clinical outcomes for patients with BRAF(V600E) melanoma, but acquired cellular resistance mediated by AKT serine/threonine kinase 1 (AKT) phosphorylation limits its efficacy. We examined the effect of resveratrol on vemurafenib-resistant melanoma cells. A vemurafenib-resistant human metastatic melanoma cell line positive for the BRAF V600E mutation was established. The anti-tumorigenic effects of vemurafenib and resveratrol, both alone and in combination, were examined through analysis of cell proliferation and protein expression. The level of phosphorylated AKT (p-AKT) was increased in the primary melanoma cells after treatment with vemurafenib, and the basal level of p-AKT was increased in vemurafenib-resistant melanoma cells. Notably, resveratrol both alone and in combination with vemurafenib effectively suppressed cell proliferation and AKT phosphorylation in both parental and vemurafenib-resistant melanoma cells. Vemurafenib resistance can be reversed by addition of resveratrol in patients undergoing treatment with BRAF inhibitors. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  15. Effects of various antineoplastic agents on an established human melanoma cell line (G-361).

    PubMed

    García Reverte, J M; Bernabeu Esclapez, A; Muñoz Ramos, J; Vicente Ortega, V; Canteras Jordana, M

    1995-01-01

    An established human melanoma cell line was treated with several concentrations of three antineoplastic drugs: melphalan (0.016, 0.032, 0.16 microns), CCNU (1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea) (0.04, 0.21, 0.42 microM), and 4-OHA (4-hydroxyanisole) (4.01 x 10(-4), 1.20 x 10(-3), 2.4 x 10(-3) microM), and the effects on cell growth and viability were compared. 24 hours after treatment, 4-OHA (ID50 = 2.4 x 10(-3) microM) was more cytotoxic than melphalan (ID50 = 0.016 microM) and CCNU (ID50 = 0.21 microM). However, after 96 hours exposure, the most effective drug was CCNU (growth rate = -1.277), which caused the death of the culture. This was followed by melphalan (growth rate = -1.024) and finally 4-OHA (growth rate = -0.69). Similar ultrastructural cell injuries were observed after the use of the three drugs: the dilation of endoplasmic reticulum vesicles and the nuclear membrane; mitochondria swelling; and the existence of lamellar structures and cytoplasmic vacuoles.

  16. NFATc2 is an intrinsic regulator of melanoma dedifferentiation.

    PubMed

    Perotti, V; Baldassari, P; Molla, A; Vegetti, C; Bersani, I; Maurichi, A; Santinami, M; Anichini, A; Mortarini, R

    2016-06-02

    Melanoma dedifferentiation, characterized by the loss of MITF and MITF regulated genes and by upregulation of stemness markers as CD271, is implicated in resistance to chemotherapy, target therapy and immunotherapy. The identification of intrinsic mechanisms fostering melanoma dedifferentiation may provide actionable therapeutic targets to improve current treatments. Here, we identify NFATc2 transcription factor as an intrinsic regulator of human melanoma dedifferentiation. In panels of melanoma cell lines, NFATc2 expression correlated inversely with MITF at both mRNA and protein levels. NFATc2(+/Hi) melanoma cell lines were CD271(+) and deficient for expression of melanocyte differentiation antigens (MDAs) MART-1, gp100, tyrosinase and of GPNMB, PGC1-α and Rab27a, all regulated by MITF. Targeting of NFATc2 by small interfering RNA, short hairpin RNA and by an NFATc2 inhibitor upregulated MITF, MDAs, GPNMB, PGC-1α, tyrosinase activity and pigmentation and suppressed CD271. Mechanistically, we found that NFATc2 controls melanoma dedifferentiation by inducing expression in neoplastic cells of membrane-bound tumor necrosis factor-α (mTNF-α) and that melanoma-expressed TNF-α regulates a c-myc-Brn2 axis. Specifically, NFATc2, mTNF-α and expression of TNF receptors were significantly correlated in panels of cell lines. NFATc2 silencing suppressed TNF-α expression, and neutralization of melanoma-expressed TNF-α promoted melanoma differentiation. Moreover, silencing of NFATc2 and TNF-α neutralization downmodulated c-myc and POU3F2/Brn2. Brn2 was strongly expressed in NFATc2(+/Hi) MITF(Lo) cell lines and its silencing upregulated MITF. Targeting of c-myc, by silencing or by a c-myc inhibitor, suppressed Brn2 and upregulated MITF and MART-1 in melanoma cells. The relevance of NFATc2-dependent melanoma dedifferentiation for immune escape was shown by cytolytic T-cell assays. NFATc2(Hi) MITF(Lo) MDA(Lo) HLA-A2.1(+) melanoma cells were poorly recognized by MDA

  17. Croton lechleri sap and isolated alkaloid taspine exhibit inhibition against human melanoma SK23 and colon cancer HT29 cell lines.

    PubMed

    Montopoli, Monica; Bertin, Riccardo; Chen, Zheng; Bolcato, Jenny; Caparrotta, Laura; Froldi, Guglielmina

    2012-12-18

    Croton lechleri Mull. Arg. (Euphorbiaceae) is a traditional medicinal plant which produces a red sap, traditionally known as "Sangre de Drago"; it is used in folk medicine externally for wounds, fractures, and haemorrhoids, internally for intestinal and stomach ulcers and also for the empirical cure of cancers. We investigated the effects of Croton lechleri sap and taspine in comparison with taxol and vinblastine on the growth of human cancer cell lines of SK23 (melanoma), LoVo and HT29 (colorectal cancer) using MTT and Trypan blue assays. Further, we studied cell cycle by flow cytometry and detected acetylated-α-tubulin by confocal microscope. Croton lechleri inhibited cell proliferation starting from 1 μg/mL in SK23 cells, whereas 10 times higher concentrations were required for growth inhibition of HT-29 and LoVo cell lines. Also taspine (0.1 μg/mL) inhibited the SK23 and HT29 cell proliferation. Further, assay was assessed on SK23 and HT29 cell lines with 24-48 h treatment with sap and taspine. Both sap and taspine inhibited cancer cell proliferation; taspine showed higher activity on SK23 cells, which was significantly increased after 48 h of SK23 treatment. Using confocal microscopy we observed that Croton lechleri (1 μg/mL) caused a loss of microtubule structure, whereas taspine (0.5 μg/mL) caused an increase in acetylated α-tubulin and a modification of cellular morphology, mainly in SK23 cells. Croton lechleri sap 10 and 50 μg/mL influence cell cycle; 50 μg/mL sap caused a dramatic reduction of cells in G(1)/G(0) and S phases with a great increase of subG(0) cells. The data showed that Croton lechleri and taspine could inhibit cell proliferation with higher potency against melanoma SK23 cells, supporting the empirical use of the sap as anticancer in ethnomedicine and taspine as a possible anticancer agent. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  18. Adoptive Cell Therapy of Melanoma with Cytokine-induced Killer Cells.

    PubMed

    Kim, Ji Sung; Kim, Yong Guk; Pyo, Minji; Lee, Hong Kyung; Hong, Jin Tae; Kim, Youngsoo; Han, Sang-Bae

    2015-04-01

    Melanoma is the most aggressive skin cancer and its incidence is gradually increasing worldwide. Patients with metastatic melanoma have a very poor prognosis (estimated 5-year survival rate of <16%). In the last few years, several drugs have been approved for malignant melanoma, such as tyrosine kinase inhibitors and immune checkpoint blockades. Although new therapeutic agents have improved progression-free and overall survival, their use is limited by drug resistance and drug-related toxicity. At the same time, adoptive cell therapy of metastatic melanoma with tumor-infiltrating lymphocytes has shown promising results in preclinical and clinical studies. In this review, we summarize the currently available drugs for treatment of malignant melanoma. In addition, we suggest cytokine-induced killer (CIK) cells as another candidate approach for adoptive cell therapy of melanoma. Our preclinical study and several previous studies have shown that CIK cells have potent anti-tumor activity against melanomas in vitro and in an in vivo human tumor xenograft model without any toxicity.

  19. Adoptive Cell Therapy of Melanoma with Cytokine-induced Killer Cells

    PubMed Central

    Kim, Ji Sung; Kim, Yong Guk; Pyo, Minji; Lee, Hong Kyung; Hong, Jin Tae; Kim, Youngsoo

    2015-01-01

    Melanoma is the most aggressive skin cancer and its incidence is gradually increasing worldwide. Patients with metastatic melanoma have a very poor prognosis (estimated 5-year survival rate of <16%). In the last few years, several drugs have been approved for malignant melanoma, such as tyrosine kinase inhibitors and immune checkpoint blockades. Although new therapeutic agents have improved progression-free and overall survival, their use is limited by drug resistance and drug-related toxicity. At the same time, adoptive cell therapy of metastatic melanoma with tumor-infiltrating lymphocytes has shown promising results in preclinical and clinical studies. In this review, we summarize the currently available drugs for treatment of malignant melanoma. In addition, we suggest cytokine-induced killer (CIK) cells as another candidate approach for adoptive cell therapy of melanoma. Our preclinical study and several previous studies have shown that CIK cells have potent anti-tumor activity against melanomas in vitro and in an in vivo human tumor xenograft model without any toxicity. PMID:25922594

  20. Radiation sensitivity of Merkel cell carcinoma cell lines

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

    Leonard, J.H.; Ramsay, J.R.; Birrell, G.W.

    1995-07-30

    Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions. Some of the MCC lines grew as suspension cultures and could not be single cell cloned; therefore, it was not possible to use clonogenic survival for all cell lines. A tetrazolium based (MTT)more » assay was used for these lines, to estimate cell growth after {gamma} irradiation. Control experiments were conducted on lymphoblastoid cell lines (LCL) and the adherent MCC line, MCC13, to demonstrate that the two assays were comparable under the conditions used. We have examined cell lines from MCC, small cell lung cancer (SCLC), malignant melanomas, Epstein Barr virus (EBV) transformed lymphocytes (LCL), and skin fibroblasts for their sensitivity to {gamma} irradiation using both clonogenic cell survival and MTT assays. The results show that the tumor cell lines have a range of sensitivities, with melanoma being more resistant (surviving fraction at 2 Gy (SF2) 0.57 and 0.56) than the small cell carcinoma lines, MCC (SF2 range 0.21-0.45, mean SF2 0.30, n = 8) and SCLC (SF2 0.31). Fibroblasts were the most sensitive (SF2 0.13-0.20, mean 0.16, n = 5). The MTT assay, when compared to clonogenic assay for the MCC13 adherent line and the LCL, gave comparable results under the conditions used. Both assays gave a range of SF2 values for the MCC cell lines, suggesting that these cancers would give a heterogeneous response in vivo. The results with the two derivative clones of MCC14 (SF2 for MCC14/1 0.38, MCC14/2 0.45) would further suggest that some of them may develop resistance during clonogenic evolution. 25 refs., 3 figs., 1 tab.« less

  1. Melanoma Stem Cells and Metastasis: Mimicking Hematopoietic Cell Trafficking?

    PubMed Central

    Lee, Nayoung; Barthel, Steven R.; Schatton, Tobias

    2014-01-01

    Malignant melanoma is a highly metastatic cancer that bears responsibility for the majority of skin cancer-related deaths. Amidst the research efforts to better understand melanoma progression, there has been increasing evidence that hints at a role for a subpopulation of virulent cancer cells, termed malignant melanoma stem or initiating cells (MMICs), in metastasis formation. MMICs are characterized by their preferential ability to initiate and propagate tumor growth and their selective capacity for self-renewal and differentiation into less tumorigenic melanoma cells. The frequency of MMICs has been shown to correlate with poor clinical prognosis in melanoma. Additionally, MMICs are enriched among circulating tumor cells (CTCs) in the peripheral blood of cancer patients, suggesting that MMICs may be a critical player in the metastatic cascade. Although these links exist between MMICs and metastatic disease, the mechanisms by which MMICs may advance metastatic progression are only beginning to be elucidated. Recent studies have shown that MMICs express molecules critical for hematopoietic cell maintenance and trafficking, providing a possible explanation for how circulating MMICs could drive melanoma dissemination. We therefore propose that MMICs might fuel melanoma metastasis by exploiting homing mechanisms commonly utilized by hematopoietic cells. Here we review the biological properties of MMICs and the existing literature on their metastatic potential. We will discuss possible mechanisms by which MMICs might initiate metastases in the context of established knowledge of cancer stem cells (CSCs) in other cancers and of hematopoietic homing molecules, with a particular focus on selectins, integrins, chemokines, and chemokine receptors known to be expressed by melanoma cells. Biological understanding of how these molecules might be utilized by MMICs to propel the metastatic cascade could critically impact the development of more effective therapies for advanced

  2. The detection of melanoma cells in peripheral blood by reverse transcription-polymerase chain reaction.

    PubMed Central

    Foss, A. J.; Guille, M. J.; Occleston, N. L.; Hykin, P. G.; Hungerford, J. L.; Lightman, S.

    1995-01-01

    Both cutaneous and uveal melanoma undergo haematogenous dissemination. Detection of tyrosinase mRNA by reverse transcription-polymerase chain reaction (RT-PCR) has been described as an extremely sensitive way of detecting circulating viable melanoma cells in the peripheral venous blood, and this technique may be of value in the early detection of dissemination. Also, it has been suggested that surgical manipulation of the eye, such as occurs during enucleation, can provoke uveal melanoma dissemination. The purpose of this study was to evaluate whether tyrosinase mRNA is detectable in the peripheral blood of patients with uveal and cutaneous melanoma and in patients with uveal melanoma undergoing surgical procedures on the eye harbouring the tumour. Venous blood samples from 36 patients diagnosed as having active uveal melanoma and from six patients with advanced metastatic cutaneous melanoma were analysed. In addition, blood samples were spiked with known numbers of cells from three cell lines and four primary uveal melanoma cultures. The reported sensitivity of the technique was confirmed, with an ability to detect down to one cell per ml of blood. All 51 blood samples from the 36 patients with uveal melanoma were negative, and this included 20 perioperative blood samples. The test was also negative for the six patients with advanced cutaneous melanoma. There were two positives among 31 control samples analysed. This study demonstrates that there are far fewer circulating viable melanocytes than has been previously supposed in patients with melanoma and that the RT-PCR is of no clinical value in detecting metastatic melanoma disease. There was no evidence for surgery causing a bolus of melanoma cells to enter the peripheral circulation. Images Figure 1 Figure 2 PMID:7599046

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

    PubMed

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

    2017-04-01

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

  4. Expression of Antimicrobial Peptides by Uveal and Cutaneous Melanoma Cells and Investigation of Their Role in Tumor Cell Migration and Vasculogenic Mimicry.

    PubMed

    Manarang, Joseph C; Otteson, Deborah C; McDermott, Alison M

    2017-11-01

    Antimicrobial peptides (AMPs) have been implicated in the pathogenesis of several cancers, although there is also evidence suggesting potential for novel, AMP-based antitumor therapies. Discerning potential roles of AMPs in tumor pathogenesis may provide valuable insight into the mechanisms of novel AMP-based antitumor therapy. mRNA expression of the AMPs α defensin (HNP-1); cathelicidin (LL-37); and β defensins (hBD-1, hBD-2, hBD-3, hBD-4) in human uveal and cutaneous melanoma cell lines, primary human uveal melanocytes, and primary human uveal melanoma cells was determined by reverse transcriptase polymerase chain reaction. An in vitro scratch assay and custom Matlab analysis were used to determine the AMP effects on melanoma cell migration. Last, the effect of specific AMPs on vasculogenic mimicry was determined by three-dimensional (3D) culture and light and fluorescence microscopy. Low-to-moderate AMP transcript levels were detected, and these varied across the cells tested. Overall, LL-37 expression was increased while hBD-4 was decreased in most melanoma cell lines, compared to primary cultured uveal melanocytes. There was no observable influence of HNP-1 and LL-37 on tumor cell migration. Additionally, aggressive cutaneous melanoma cells grown in 3D cultures exhibited vasculogenic mimicry, although AMP exposure did not alter this process. Collectively, our data show that although AMP mRNA expression is variable between uveal and cutaneous melanoma cells, these peptides have little influence on major characteristics that contribute to tumor aggressiveness and progression.

  5. Rb-Raf-1 interaction disruptor RRD-251 induces apoptosis in metastatic melanoma cells and synergizes with dacarbazine

    PubMed Central

    Singh, Sandeep; Davis, Rebecca; Alamanda, Vignesh; Pireddu, Roberta; Pernazza, Daniel; Sebti, Said; Lawrence, Nicholas; Chellappan, Srikumar

    2010-01-01

    Metastatic melanoma is an aggressive cancer with very low response rate against conventional chemotherapeutic agents such as dacarbazine (DTIC). Inhibitor of Rb-Raf-1 interaction (RRD-251) was tested against the melanoma cell lines SK-MEL-28, SK-MEL-5 and SK-MEL-2. RRD-251 was found to be a potent inhibitor of melanoma cell proliferation, irrespective of V600E B-Raf mutation status of the cell lines. In a SK-MEL-28 xenograft experiment, RRD-251 exerted a significant suppression of tumor growth compared to vehicle (p=0.003). Similar to in vitro effects, tumors from RRD-251 treated animals showed decreased Rb-Raf-1 interaction in vivo. Growth suppressive effects of RRD-251 were associated with induction of apoptosis as well as a G1 arrest, with an accompanying decrease in S-phase cells. RRD-251 inhibited Rb phosphorylation, and downregulated E2F1 protein levels in these cells. Real-time PCR analysis showed that RRD-251 caused downregulation of cell cycle regulatory genes thymidylate synthase (TS) and cdc6 as well as anti-apoptotic gene Mcl-1. Combinatorial treatment of RRD-251 and DTIC resulted in a significantly higher apoptosis in DTIC resistant cell lines SK-MEL-28 and SK-MEL-5, as revealed by increased Caspase-3 activity and PARP cleavage. Since aberrant Rb/E2F pathway is associated with melanoma progression and resistance to apoptosis, these results suggest that the Rb-Raf-1 inhibitor could be an effective agent for melanoma treatment, either alone or in combination with DTIC. PMID:21139044

  6. Rb-Raf-1 interaction disruptor RRD-251 induces apoptosis in metastatic melanoma cells and synergizes with dacarbazine.

    PubMed

    Singh, Sandeep; Davis, Rebecca; Alamanda, Vignesh; Pireddu, Roberta; Pernazza, Daniel; Sebti, Said; Lawrence, Nicholas; Chellappan, Srikumar

    2010-12-01

    Metastatic melanoma is an aggressive cancer with very low response rate against conventional chemotherapeutic agents such as dacarbazine (DTIC). Inhibitor of Rb-Raf-1 interaction RRD-251 was tested against the melanoma cell lines SK-MEL-28, SK-MEL-5, and SK-MEL-2. RRD-251 was found to be a potent inhibitor of melanoma cell proliferation, irrespective of V600E B-Raf mutation status of the cell lines. In a SK-MEL-28 xenograft experiment, RRD-251 exerted a significant suppression of tumor growth compared with vehicle (P = 0.003). Similar to in vitro effects, tumors from RRD-251-treated animals showed decreased Rb-Raf-1 interaction in vivo. Growth suppressive effects of RRD-251 were associated with induction of apoptosis as well as a G(1) arrest, with an accompanying decrease in S-phase cells. RRD-251 inhibited Rb phosphorylation and downregulated E2F1 protein levels in these cells. Real-time PCR analysis showed that RRD-251 caused downregulation of cell-cycle regulatory genes thymidylate synthase (TS) and cdc6 as well as the antiapoptotic gene Mcl-1. Combinatorial treatment of RRD-251 and DTIC resulted in a significantly higher apoptosis in DTIC resistant cell lines SK-MEL-28 and SK-MEL-5, as revealed by increased caspase-3 activity and PARP cleavage. Because aberrant Rb/E2F pathway is associated with melanoma progression and resistance to apoptosis, these results suggest that the Rb-Raf-1 inhibitor could be an effective agent for melanoma treatment, either alone or in combination with DTIC. ©2010 AACR.

  7. Adoptive Cell Therapy for Metastatic Melanoma.

    PubMed

    Merhavi-Shoham, Efrat; Itzhaki, Orit; Markel, Gal; Schachter, Jacob; Besser, Michal J

    Adoptive cell therapy (ACT) of tumor-infiltrating lymphocytes (TILs) is a powerful form of immunotherapy by inducing durable complete responses that significantly extend the survival of melanoma patients. Mutation-derived neoantigens were recently identified as key factors for tumor recognition and rejection by TILs. The isolation of T-cell receptor (TCR) genes directed against neoantigens and their retransduction into peripheral T cells may provide a new form of ACT.Genetic modifications of T cells with chimeric antigen receptors (CARs) have demonstrated remarkable clinical results in hematologic malignancies, but are so far less effective in solid tumors. Only very limited reports exist in melanoma. Progress in CAR T-cell engineering, including neutralization of inhibitory signals or additional safety switches, may open opportunities also in melanoma.We review clinical results and latest developments of adoptive therapies with TILs, T-cell receptor, and CAR-modified T cells and discuss future directions for the treatment of melanoma.

  8. Fasting boosts sensitivity of human skin melanoma to cisplatin-induced cell death

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

    Antunes, Fernanda; Corazzari, Marco; National Institute for Infectious Diseases IRCCS “Lazzaro Spallanzani”

    Melanoma is one of leading cause of tumor death worldwide. Anti-cancer strategy includes combination of different chemo-therapeutic agents as well as radiation; however these treatments have limited efficacy and induce significant toxic effects on healthy cells. One of most promising novel therapeutic approach to cancer therapy is the combination of anti-cancer drugs with calorie restriction. Here we investigated the effect Cisplatin (CDDP), one of the most potent chemotherapeutic agent used to treat tumors, in association with fasting in wild type and mutated BRAF{sup V600E} melanoma cell lines. Here we show that nutrient deprivation can consistently enhance the sensitivity of tumormore » cells to cell death induction by CDDP, also of those malignancies particularly resistant to any treatment, such as oncogenic BRAF melanomas. Mechanistic studies revealed that the combined therapy induced cell death is characterized by ROS accumulation and ATF4 in the absence of ER-stress. In addition, we show that autophagy is not involved in the enhanced sensitivity of melanoma cells to combined CDDP/EBSS-induced apoptosis. While, the exposure to 2-DG further enhanced the apoptotic rate observed in SK Mel 28 cells upon treatment with both CDDP and EBSS. - Highlights: • Calorie restriction associated to chemo-therapeutic drugs enhance cell death induction in many resistant malignancies • Cisplatin in association with starvation significantly increases cell death also in those high resistant melanoma cells bearing BRAF mutations • Combined treatment also including 2-DG results in similar cell death levels in both wild type and mutated BRAF cells.« less

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

    PubMed

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

    2018-03-04

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

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

    PubMed

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

    2010-05-01

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

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

    PubMed Central

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

    2010-01-01

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

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

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

    Wang, Dong; The 309th Hospital of China People's Liberation Army, Beijing 100091; Wang, Junyun

    2016-05-13

    MAGE-A1 belongs to the chromosome X-clustered genes of cancer-testis antigen family and is normally expressed in the human germ line but is also overexpressed in various tumors. Previous studies of MAGE-A1 in melanoma mainly focused on methylation changes or its role in immunotherapy, however, its biological functions in melanoma have remained unknown. In order to determine the role of MAGE-A1 in melanoma growth and metastasis, we manipulated melanoma cell lines with overexpression and knockdown of MAGE-A1. Integration of cell proliferation assays, transwell migration and invasion assays, and RNA-Seq analysis revealed that up-regulation of MAGE-A1 dramatically promoted proliferation, migration, and invasionmore » of human melanoma cell lines in vitro, while down-regulation of MAGE-A1 inhibited those characteristics associated with tumor cells. Furthermore, transcriptome sequencing revealed that MAGE-A1 exerts its tumor promoting activity by activating p-C-JUN directly or through ERK-MAPK signaling pathways. Based on our findings, we propose that MAGE-A1 may be a potential therapeutic target for melanoma patients. - Highlights: • MAGE-A1 promotes proliferation and clone formation in melanoma cell lines. • MAGE-A1 enhances tumor cell migration and invasion in melanoma cell lines. • Network including C-JUN, IL8, and ARHGAP29 play critical role in malignant melanoma. • Oncogenic MAGE-A1 increases p-C-JUN levels, possibly via ERK-MAPK signaling pathway.« less

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

    PubMed

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

    2011-02-15

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

  14. Transketolase-like 1 ectopic expression is associated with DNA hypomethylation and induces the Warburg effect in melanoma cells.

    PubMed

    Jayachandran, Aparna; Lo, Pu-Han; Chueh, Anderly C; Prithviraj, Prashanth; Molania, Ramyar; Davalos-Salas, Mercedes; Anaka, Matthew; Walkiewicz, Marzena; Cebon, Jonathan; Behren, Andreas

    2016-02-22

    The metabolism of cancer cells is often reprogrammed by dysregulation of metabolic enzymes. Transketolase-like 1 (TKTL1) is a homodimeric transketolase linking the pentose-phosphate pathway with the glycolytic pathway. It is generally silenced at a transcriptional level in somatic tissues. However, in human cancers its expression is associated with the acquisition of a glycolytic phenotype (the Warburg effect) by cancer cells that contributes to the progression of malignant tumors. In melanoma, defective promoter methylation results in the expression of genes and their products that can affect the tumor cell's phenotype including the modification of immune and functional characteristics. The present study evaluates the role of TKTL1 as a mediator of disease progression in melanoma associated with a defective methylation phenotype. The expression of TKTL1 in metastatic melanoma tumors and cell lines was analysed by qRT-PCR and immunohistochemistry. The promoter methylation status of TKTL1 in melanoma cells was evaluated by quantitative methylation specific PCR. Using qRT-PCR, the effect of a DNA demethylating agent 5-aza-2'-deoxycytidine (5aza) on the expression of TKTL1 was examined. Biochemical and molecular analyses such as glucose consumption, lactate production, invasion, proliferation and cell cycle progression together with ectopic expression and siRNA mediated knockdown were used to investigate the role of TKTL1 in melanoma cells. Expression of TKTL1 was highly restricted in normal adult tissues and was overexpressed in a subset of metastatic melanoma tumors and derived cell lines. The TKTL1 promoter was activated by hypomethylation and treatment with 5aza induced TKTL1 expression in melanoma cells. Augmented expression of TKTL1 in melanoma cells was associated with a glycolytic phenotype. Loss and gain of function studies revealed that TKTL1 contributed to enhanced invasion of melanoma cells. Our data provide evidence for an important role of TKTL1 in

  15. Metastatic melanoma: results of 'classical' second-line treatment with cytotoxic chemotherapies.

    PubMed

    Perrin, Christophe; Pracht, Marc; Talour, Karen; Adamski, Henri; Cumin, Isabelle; Porneuf, Marc; Talarmin, Marie; Mesbah, Habiba; Audrain, Odile; Moignet, Aline; Lefeuvre-Plesse, Claudia; Lesimple, Thierry

    2014-10-01

    Metastatic melanoma is one of the most aggressive tumours, with a median survival that does not exceed 12 months. None of the cytotoxic first-line therapies have shown survival benefit in randomised clinical trials. To describe clinical benefit of second-line cytotoxic chemotherapy in the second line of treatment for metastatic melanoma. In a retrospective study, we analyse the outcome of patients with metastatic melanoma who had received two lines or more of cytotoxic treatments in four French dermato-oncology departments between 1999 and 2009. We describe the outcomes for 109 patients. Most of these patients received dacarbazine for the first line of chemotherapy and fotemustine for the second line of chemotherapy (67.0 and 64.2%, respectively). A clinical benefit was observed in 24.1% of the patients and overall survival was 4.1 months after the second-line treatment. At least 23.8% of patients suffered from grade 3 or 4 toxicities. The presence of more than two sites of metastasis and an M1c staging according to the AJCC classification represented negative predictive factors of clinical benefit. This study shows the modest benefit of a second line of cytotoxic chemotherapy in a nonselected population. If eligible, these patients should be proposed for ongoing clinical trials or for targeted therapies.

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

    PubMed Central

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

    2011-01-01

    Notoriously chemoresistant melanoma has become the most prevalent form of cancer for the 25–29 North American age demographic. Standard treatment after early detection involves surgical excision (recurrence is possible), and metastatic melanoma is refractory to immuno-, radio-, and most harmful chemotherapies. Various natural compounds have shown efficacy in killing different cancers, albeit not always specifically. In this study, we show that dandelion root extract (DRE) specifically and effectively induces apoptosis in human melanoma cells without inducing toxicity in noncancerous cells. Characteristic apoptotic morphology of nuclear condensation and phosphatidylserine flipping to the outer leaflet of the plasma membrane of A375 human melanoma cells was observed within 48 hours. DRE-induced apoptosis activates caspase-8 in A375 cells early on, demonstrating employment of an extrinsic apoptotic pathway to kill A375 cells. Reactive Oxygen Species (ROS) generated from DRE-treated isolated mitochondria indicates that natural compounds in DRE can also directly target mitochondria. Interestingly, the relatively resistant G361 human melanoma cell line responded to DRE when combined with the metabolism interfering antitype II diabetic drug metformin. Therefore, treatment with this common, yet potent extract of natural compounds has proven novel in specifically inducing apoptosis in chemoresistant melanoma, without toxicity to healthy cells. PMID:21234313

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

    PubMed

    Devarapu, Satish Kumar; Mamidi, Srinivas; Plöger, Frank; Dill, Othmar; Blixt, Ola; Kirschfink, Michael; Schwartz-Albiez, Reinhard

    2016-06-15

    A small percentage of healthy donors identified in the Western population carry antibodies in their peripheral blood which convey cytotoxic activity against certain human melanoma and neuroblastoma cell lines. We measured the cytotoxic activity of sera and plasmas from healthy donors on the human neuroblastoma cell line Kelly and various melanoma cell lines. Antibodies of IgM isotype, presumably belonging to the class of naturally occurring antibodies, exerted cytotoxic activity in a complement-dependent fashion. Apart from complement-dependent tumor cell lysis, we observed C3 opsonization in all tumor cell lines upon treatment with cytotoxic plasmas. Cell lines tested primarily expressed membrane complement regulatory proteins (mCRP) CD46, CD55 and CD59 to various extents. Blocking of mCRPs by monoclonal antibodies enhanced cell lysis and opsonization, though some melanoma cells remained resistant to complement attack. Epitopes recognized by cytotoxic antibodies were represented by gangliosides such as GD2 and GD3, as evidenced by cellular sialidase pretreatment and enhanced expression of distinct gangliosides. It remains to be clarified why only a small fraction of healthy persons carry these antitumor cytotoxic antibodies. © 2016 UICC.

  18. PD-L1 Promotes Self-Renewal and Tumorigenicity of Malignant Melanoma Initiating Cells

    PubMed Central

    Dang, Jianzhong; Zha, Hui; Zhang, Bingyu; Lin, Ming

    2017-01-01

    Recent studies have indicated that therapeutic antibodies targeting PD-L1 show remarkable efficacy in clinical trials in multiple tumors and that a melanoma cell-intrinsic PD-1: PD-L1 axis promotes tumor growth. However, few studies have shown tumor-intrinsic PD-L1 effects in malignant melanoma initiating cells (MMICs). Here, we aim to determine the possible regulatory effects of PD-L1 on MMICs. The ALDEFLUOR kit was used to identify ALDH+ MMICs. Flow cytometry was used to examine the expression of PD-L1 on ALDH+ MMICs. To determine the role of PD-L1 in MMICs self-renewal, we cultured melanoma cells with anti-PD-L1 and measured tumorsphere formation and apoptosis. In addition, the effects of anti-PD-L1 on tumorigenicity and residual ALDH+ MMICs in tumors were evaluated in vivo. We demonstrated that melanoma cell-intrinsic PD-L1 was expressed in ALDH+ MMICs. Blocking PD-L1 in melanoma cell lines impaired tumorsphere formation and induced the apoptosis of sphere cells. In addition, blocking PD-L1 inhibited tumor growth in vivo. We observed residual ALDH+ MMICs within the tumor. The results showed that blocking PD-L1 also significantly decreased the residual ALDH+ MMICs in the tumors. In conclusion, these results suggest a new mechanism underlying melanoma progression and PD-L1-targeted therapy, which is distinct from the immunomodulatory actions of PD-L1. PMID:29250533

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

    PubMed Central

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

    2004-01-01

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

  20. Quantitative comparison of the spreading and invasion of radial growth phase and metastatic melanoma cells in a three-dimensional human skin equivalent model.

    PubMed

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

    2017-01-01

    Standard two-dimensional (2D) cell migration assays do not provide information about vertical invasion processes, which are critical for melanoma progression. We provide information about three-dimensional (3D) melanoma cell migration, proliferation and invasion in a 3D melanoma skin equivalent (MSE) model. In particular, we pay careful attention to compare the structure of the tissues in the MSE with similarly-prepared 3D human skin equivalent (HSE) models. The HSE model is identically prepared to the MSE model except that melanoma cells are omitted. Using the MSE model, we examine melanoma migration, proliferation and invasion from two different human melanoma cell lines. One cell line, WM35, is associated with the early phase of the disease where spreading is thought to be confined to the epidermis. The other cell line, SK-MEL-28, is associated with the later phase of the disease where spreading into the dermis is expected. 3D MSE and HSE models are constructed using human de-epidermised dermis (DED) prepared from skin tissue. Primary fibroblasts and primary keratinocytes are used in the MSE and HSE models to ensure the formation of a stratified epidermis, with a well-defined basement membrane. Radial spreading of cells across the surface of the HSE and MSE models is observed. Vertical invasion of melanoma cells downward through the skin is observed and measured using immunohistochemistry. All measurements of invasion are made at day 0, 9, 15 and 20, providing detailed time course data. Both HSE and MSE models are similar to native skin in vivo , with a well-defined stratification of the epidermis that is separated from the dermis by a basement membrane. In the HSE and MSE we find fibroblast cells confined to the dermis, and differentiated keratinocytes in the epidermis. In the MSE, melanoma cells form colonies in the epidermis during the early part of the experiment. In the later stage of the experiment, the melanoma cells in the MSE invade deeper into the

  1. Quantitative comparison of the spreading and invasion of radial growth phase and metastatic melanoma cells in a three-dimensional human skin equivalent model

    PubMed Central

    Haridas, Parvathi; McGovern, Jacqui A.; McElwain, Sean D.L.

    2017-01-01

    Background Standard two-dimensional (2D) cell migration assays do not provide information about vertical invasion processes, which are critical for melanoma progression. We provide information about three-dimensional (3D) melanoma cell migration, proliferation and invasion in a 3D melanoma skin equivalent (MSE) model. In particular, we pay careful attention to compare the structure of the tissues in the MSE with similarly-prepared 3D human skin equivalent (HSE) models. The HSE model is identically prepared to the MSE model except that melanoma cells are omitted. Using the MSE model, we examine melanoma migration, proliferation and invasion from two different human melanoma cell lines. One cell line, WM35, is associated with the early phase of the disease where spreading is thought to be confined to the epidermis. The other cell line, SK-MEL-28, is associated with the later phase of the disease where spreading into the dermis is expected. Methods 3D MSE and HSE models are constructed using human de-epidermised dermis (DED) prepared from skin tissue. Primary fibroblasts and primary keratinocytes are used in the MSE and HSE models to ensure the formation of a stratified epidermis, with a well-defined basement membrane. Radial spreading of cells across the surface of the HSE and MSE models is observed. Vertical invasion of melanoma cells downward through the skin is observed and measured using immunohistochemistry. All measurements of invasion are made at day 0, 9, 15 and 20, providing detailed time course data. Results Both HSE and MSE models are similar to native skin in vivo, with a well-defined stratification of the epidermis that is separated from the dermis by a basement membrane. In the HSE and MSE we find fibroblast cells confined to the dermis, and differentiated keratinocytes in the epidermis. In the MSE, melanoma cells form colonies in the epidermis during the early part of the experiment. In the later stage of the experiment, the melanoma cells in the MSE

  2. VEGFR-1 expressed by malignant melanoma-initiating cells is required for tumor growth.

    PubMed

    Frank, Natasha Y; Schatton, Tobias; Kim, Soo; Zhan, Qian; Wilson, Brian J; Ma, Jie; Saab, Karim R; Osherov, Veronika; Widlund, Hans R; Gasser, Martin; Waaga-Gasser, Ana-Maria; Kupper, Thomas S; Murphy, George F; Frank, Markus H

    2011-02-15

    Melanoma growth is driven by malignant melanoma-initiating cells (MMIC) identified by expression of the ATP-binding cassette (ABC) member ABCB5. ABCB5(+) melanoma subpopulations have been shown to overexpress the vasculogenic differentiation markers CD144 (VE-cadherin) and TIE1 and are associated with CD31(-) vasculogenic mimicry (VM), an established biomarker associated with increased patient mortality. Here we identify a critical role for VEGFR-1 signaling in ABCB5(+) MMIC-dependent VM and tumor growth. Global gene expression analyses, validated by mRNA and protein determinations, revealed preferential expression of VEGFR-1 on ABCB5(+) tumor cells purified from clinical melanomas and established melanoma lines. In vitro, VEGF induced the expression of CD144 in ABCB5(+) subpopulations that constitutively expressed VEGFR-1 but not in ABCB5(-) bulk populations that were predominantly VEGFR-1(-). In vivo, melanoma-specific shRNA-mediated knockdown of VEGFR-1 blocked the development of ABCB5(+) VM morphology and inhibited ABCB5(+) VM-associated production of the secreted melanoma mitogen laminin. Moreover, melanoma-specific VEGFR-1 knockdown markedly inhibited tumor growth (by > 90%). Our results show that VEGFR-1 function in MMIC regulates VM and associated laminin production and show that this function represents one mechanism through which MMICs promote tumor growth. ©2011 AACR.

  3. WNT5A enhances resistance of melanoma cells to targeted BRAF inhibitors

    PubMed Central

    Anastas, Jamie N.; Kulikauskas, Rima M.; Tamir, Tigist; Rizos, Helen; Long, Georgina V.; von Euw, Erika M.; Yang, Pei-Tzu; Chen, Hsiao-Wang; Haydu, Lauren; Toroni, Rachel A.; Lucero, Olivia M.; Chien, Andy J.; Moon, Randall T.

    2014-01-01

    About half of all melanomas harbor a mutation that results in a constitutively active BRAF kinase mutant (BRAFV600E/K) that can be selectively inhibited by targeted BRAF inhibitors (BRAFis). While patients treated with BRAFis initially exhibit measurable clinical improvement, the majority of patients eventually develop drug resistance and relapse. Here, we observed marked elevation of WNT5A in a subset of tumors from patients exhibiting disease progression on BRAFi therapy. WNT5A transcript and protein were also elevated in BRAFi-resistant melanoma cell lines generated by long-term in vitro treatment with BRAFi. RNAi-mediated reduction of endogenous WNT5A in melanoma decreased cell growth, increased apoptosis in response to BRAFi challenge, and decreased the activity of prosurvival AKT signaling. Conversely, overexpression of WNT5A promoted melanoma growth, tumorigenesis, and activation of AKT signaling. Similarly to WNT5A knockdown, knockdown of the WNT receptors FZD7 and RYK inhibited growth, sensitized melanoma cells to BRAFi, and reduced AKT activation. Together, these findings suggest that chronic BRAF inhibition elevates WNT5A expression, which promotes AKT signaling through FZD7 and RYK, leading to increased growth and therapeutic resistance. Furthermore, increased WNT5A expression in BRAFi-resistant melanomas correlates with a specific transcriptional signature, which identifies potential therapeutic targets to reduce clinical BRAFi resistance. PMID:24865425

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

    PubMed

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

    2016-03-01

    Rose Bengal (RB) is an anionic water-soluble xanthene dye, which used for many years to assess eye cornea and conjunctiva damage. RB showed strong absorption maxima (λmax) under visible light followed by UV-B and UV-A. RB under sunlight exposure showed a time-dependent photodegradation. Our results show that photosensitized RB generates (1)O2 via Type-II photodynamic pathway and induced DNA damage under sunlight/UV-R exposure. 2'dGuO degradation, micronuclei formation, and single- and double-strand breakage were the outcome of photogenotoxicity caused by RB. Quenching studies with NaN3 advocate the involvement of (1)O2 in RB photogenotoxicity. RB induced linoleic acid photoperoxidation, which was parallel to (1)O2-mediated DNA damage. Oxidative stress in A375 cell line (human melanoma cell line) was detected through DCF-DA assay. Photosensitized RB decreased maximum cellular viability under sunlight followed by UV-B and UV-A exposures. Apoptosis was detected as a pattern of cell death through the increased of caspase-3 activity, decreased mitochondrial membrane potential, and PS translocation through inner to outer plasma membrane. Increased cytosolic levels of Bax also advocate the apoptotic cell death. We propose a p53-mediated apoptosis via increased expression of Bax gene and protein. Thus, the exact mechanism behind RB phototoxicity was the involvement of (1)O2, which induced oxidative stress-mediated DNA and membrane damage, finally apoptotic cell death under natural sunlight exposure. The study suggests that after the use of RB, sunlight exposure may avoid to prevent from its harmful effects. Copyright © 2015. Published by Elsevier B.V.

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

    PubMed

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

    2017-02-23

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

  6. Differential chemosensitivity to antifolate drugs between RAS and BRAF melanoma cells

    PubMed Central

    2014-01-01

    Background The importance of the genetic background of cancer cells for the individual susceptibility to cancer treatments is increasingly apparent. In melanoma, the existence of a BRAF mutation is a main predictor for successful BRAF-targeted therapy. However, despite initial successes with these therapies, patients relapse within a year and have to move on to other therapies. Moreover, patients harbouring a wild type BRAF gene (including 25% with NRAS mutations) still require alternative treatment such as chemotherapy. Multiple genetic parameters have been associated with response to chemotherapy, but despite their high frequency in melanoma nothing is known about the impact of BRAF or NRAS mutations on the response to chemotherapeutic agents. Methods Using cell proliferation and DNA methylation assays, FACS analysis and quantitative-RT-PCR we have characterised the response of a panel of NRAS and BRAF mutant melanoma cell lines to various chemotherapy drugs, amongst them dacarbazine (DTIC) and temozolomide (TMZ) and DNA synthesis inhibitors. Results Although both, DTIC and TMZ act as alkylating agents through the same intermediate, NRAS and BRAF mutant cells responded differentially only to DTIC. Further analysis revealed that the growth-inhibitory effects mediated by DTIC were rather due to interference with nucleotide salvaging, and that NRAS mutant melanoma cells exhibit higher activity of the nucleotide synthesis enzymes IMPDH and TK1. Importantly, the enhanced ability of RAS mutant cells to use nucleotide salvaging resulted in resistance to DHFR inhibitors. Conclusion In summary, our data suggest that the genetic background in melanoma cells influences the response to inhibitors blocking de novo DNA synthesis, and that defining the RAS mutation status could be used to stratify patients for the use of antifolate drugs. PMID:24941944

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

    PubMed Central

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

    2010-01-01

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

  8. MiR-135 post-transcriptionally regulates FOXO1 expression and promotes cell proliferation in human malignant melanoma cells.

    PubMed

    Ren, Jian-Wen; Li, Zhang-Jun; Tu, Chen

    2015-01-01

    Malignant melanoma is the deadliest form of all skin cancers. Recently, microRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression by targeted repression of transcription and translation and play essential roles during cancer development. Our study showed that miR-135a is upregulated in malignant melanoma tissues and cell lines by using Real-time PCR assay. Enforced expression of miR-135a in malignant melanoma cells promotes cell proliferation, tumorigenicity, and cell cycle progression, whereas inhibition of miR-135a reverses the function. Additionally, we demonstrated FOXO1 is a direct target of miR-135a and transcriptionally down-regulated by miR-135a. Ectopic expression of miR-135a led to downregulation of the FOXO1 protein, resulting in upregulation of Cyclin D1, and downregulation of P21(Cip1) and P27(Kip1) through AKT pathway. Our findings suggested that miR-135a represents a potential onco-miRNA and plays an important role in malignant melanoma progression by suppressing FOXO1 expression.

  9. Inhibition of melanoma cell proliferation by resveratrol is correlated with upregulation of quinone reductase 2 and p53

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

    Hsieh Tzechen; Wang Zhirong; Hamby, Carl V.

    2005-08-19

    Resveratrol (trans-3,4',5-trihydroxystilbene) is a grape-derived polyphenol under intensive study for its potential in cancer prevention. In the case of cultured human melanoma cells, no one to our knowledge has investigated whether resveratrol exerts similar anti-proliferative activities in cells with different metastatic potential. Therefore, we examined the effects of this polyphenol on the growth of weakly metastatic Line IV clone 3 and on autologous, highly metastatic Line IV clone 1 cultured melanoma cells. Comparable inhibition of growth and colony formation resulted from treatment by resveratrol in both cell lines. Flow cytometric analysis revealed that resveratrol-treated clone 1 cells had a dose-dependentmore » increase in S phase and a concomitant reduction in the G{sub 1} phase. No detectable change in cell cycle phase distribution was found in similarly treated clone 3 cells. Western blots demonstrated a significant increase in the expression of the tumor suppressor gene p53, without a commensurate change in p21 and several other cell cycle regulatory proteins in both cell types. Chromatography of Line IV clone 3 and clone 1 cell extracts on resveratrol affinity columns revealed that the basal expression of dihydronicotinamide riboside quinone reductase 2 (NQO2) was higher in Line IV clone 1 than clone 3 cells. Levels of NQO2 but not its structural analog NQO1 were dose-dependently increased by resveratrol in both cell lines. We propose that induction of NQO2 may relate to the observed increased expression of p53 that, in turn, contributes to the observed suppression of cell growth in both melanoma cell lines.« less

  10. AIM1 and LINE-1 Epigenetic Aberrations In Tumor and Serum Relate to Melanoma Progression and Disease Outcome

    PubMed Central

    Hoshimoto, Sojun; Kuo, Christine; Chong, Kelly; Takeshima, Ling; Takei, Yoshiki; Li, Michelle; Huang, Sharon; Sim, Myung-Shin; Morton, Donald L.; Hoon, Dave S.B.

    2012-01-01

    Aberrations in the methylation status of non-coding genomic repeat DNA sequences and specific gene promoter region are important epigenetic events in melanoma progression. Promoter methylation status in LINE-1 and Absent in melanoma-1(AIM1;6q21) associated with melanoma progression and disease outcome was assessed. LINE-1 and AIM1 methylation status was assessed in paraffin-embedded archival tissues(PEAT)(n=133) and melanoma patients’ serum(n=56). LINE-1 U-Index(hypomethylation) and AIM1 were analyzed in microdissected melanoma PEAT sections. The LINE-1 U-Index of melanoma(n=100) was significantly higher than that of normal skin(n=14) and nevi(n=12)(P=0.0004). LINE-1 U-Index level was elevated with increasing AJCC stage(P<0.0001). AIM1 promoter hypermethylation was found in higher frequency(P=0.005) in metastatic melanoma(65%) than in primary melanomas(38%). When analyzed, high LINE-1 U-Index and/or AIM1 methylation in melanomas were associated with disease-free survival(DFS) and overall survival(OS) in Stage I/II patients (P=0.017, 0.027; respectively). In multivariate analysis, melanoma AIM1 methylation status was a significant prognostic factor of OS(P=0.032). Furthermore, serum unmethylated LINE-1 was at higher levels in both stage III(n=20) and stage IV(n=36) patients compared to healthy donors(n=14)(P=0.022). Circulating methylated AIM1 was detected in patients’ serum and was predictive of OS in Stage IV patients (P=0.009). LINE-1 hypomethylation and AIM1 hypermethylation have prognostic utility in both melanoma patients’ tumors and serum. PMID:22402438

  11. Multiple Isoforms of ANRIL in Melanoma Cells: Structural Complexity Suggests Variations in Processing.

    PubMed

    Sarkar, Debina; Oghabian, Ali; Bodiyabadu, Pasani K; Joseph, Wayne R; Leung, Euphemia Y; Finlay, Graeme J; Baguley, Bruce C; Askarian-Amiri, Marjan E

    2017-06-27

    The long non-coding RNA ANRIL , antisense to the CDKN2B locus, is transcribed from a gene that encompasses multiple disease-associated polymorphisms. Despite the identification of multiple isoforms of ANRIL , expression of certain transcripts has been found to be tissue-specific and the characterisation of ANRIL transcripts remains incomplete. Several functions have been associated with ANRIL . In our judgement, studies on ANRIL functionality are premature pending a more complete appreciation of the profusion of isoforms. We found differential expression of ANRIL exons, which indicates that multiple isoforms exist in melanoma cells. In addition to linear isoforms, we identified circular forms of ANRIL ( circANRIL ). Further characterisation of circANR IL in two patient-derived metastatic melanoma cell lines (NZM7 and NZM37) revealed the existence of a rich assortment of circular isoforms. Moreover, in the two melanoma cell lines investigated, the complements of circANRIL isoforms were almost completely different. Novel exons were also discovered. We also found the family of linear ANRIL was enriched in the nucleus, whilst the circular isoforms were enriched in the cytoplasm and they differed markedly in stability. With respect to the variable processing of circANRIL species, bioinformatic analysis indicated that intronic Arthrobacter luteus (Alu) restriction endonuclease inverted repeats and exon skipping were not involved in selection of back-spliced exon junctions. Based on our findings, we hypothesise that " ANRIL " has wholly distinct dual sets of functions in melanoma. This reveals the dynamic nature of the locus and constitutes a basis for investigating the functions of ANRIL in melanoma.

  12. Effects of SASH1 on melanoma cell proliferation and apoptosis in vitro.

    PubMed

    Lin, Sheyu; Zhang, Junyu; Xu, Jiawei; Wang, Honglian; Sang, Qing; Xing, Qinghe; He, Lin

    2012-12-01

    The SAM and SH3 domain containing 1 (SASH1) gene was originally identified as a potential tumor suppressor gene in breast cancer, mapped on chromosome 6q24.3. The expression of SASH1 plays a prognostic role in human colon cancer. Its expression is frequently downregulated in several human malignancies. However, the biological function of SASH1 in melanoma cells is yet to be determined. In this study, in order to investigate the tumor suppressive effects of the SASH1 gene, an A-375 stable melanoma cell line was established, overexpressing the SASH1 gene. The stable cell line was examined using proliferation assay, apoptosis assay, cell cycle analysis and real-time PCR. The results indicated that the tumor suppressive activity of SASH1 derived from G2/M arrest in A-375 cells, and that the phosphorylation of Cdc2 or the disruption of cyclin B-Cdc2 binding may be responsible for the G2/M arrest.

  13. Regulator of G protein signaling 4 inhibits human melanoma cells proliferation and invasion through the PI3K/AKT signaling pathway

    PubMed Central

    Xue, Xiaotong; Wang, Lihua; Meng, Xianguang; Jiao, Jing; Dang, Ningning

    2017-01-01

    Melanoma is a tumor produced by skin melanocytes, which has a high metastatic rate and poor prognosis. So far, plenty of work has been done on melanoma, but mechanisms underlying melanoma development have not been fully elucidated. Here we identified regulator of G protein signaling 4(RGS4) as novel therapeutic target for malignant melanoma and its regulating effect on melanoma. We found that endogenous RGS4 expression was much lower in melanoma tissues and cells. In A375 cell line with low endogenous RGS4 expression, the function of RGS4 was detected by up-regulation its expression with pcDNA3.1-RGS4 and knockdown its expression with siRNA. Our results showed that RGS4 could significantly reduce the proliferation, migration and invasion of melanoma cells. RGS4 is an important regulator for the apoptosis of melanocyte, and the apoptosis rate is significantly decreased in low RGS4 enviroment. RGS4 induced non-activation of PI3K/AKT pathway, resulting in decreased expression of E2F1 and Cyclin D1, thus constraining cell proliferation and invasion. These results were further confirmed in M14 cell lines. Collectively, our findings show that RGS4 plays an important role in multiple cellular functions of melanoma development and is valuable to be a therapeutic target. PMID:29108247

  14. The helicase HAGE expressed by malignant melanoma-initiating cells is required for tumor cell proliferation in vivo.

    PubMed

    Linley, Adam J; Mathieu, Morgan G; Miles, Amanda K; Rees, Robert C; McArdle, Stephanie E B; Regad, Tarik

    2012-04-20

    Malignant melanoma-initiating cells (MMIC) are a subpopulation of cells responsible for melanoma tumor growth and progression. They are defined by the expression of the ATP-binding cassette (ABC) subfamily B member 5 (ABCB5). Here, we identified a critical role for the DEAD-box helicase antigen (HAGE) in ABCB5+ MMIC-dependent tumorigenesis and show that HAGE-specific inactivation inhibits melanoma tumor growth mediated by this tumor-initiating population. Knockdown of HAGE led to a significant decrease in RAS protein expression with a concomitant decrease in activation of the AKT and ERK signaling pathways implicated to play an important role in melanoma progression. To confirm that the reduction in NRAS (Neuroblastoma RAS) expression was dependent on the HAGE helicase activity, we showed that NRAS, effectively silenced by siRNA, could be rescued by reintroduction of HAGE in cells lacking HAGE. Furthermore, we provide a mechanism by which HAGE promotes NRAS unwinding in vitro. We also observed using tumor transplantation in Non-obese diabetic/severe combined immunodeficiency mice that the HAGE knockdown in a ABCB5+ melanoma cell line displayed a significant decrease in tumor growth and compared with the control. Our results suggest that the helicase HAGE is required for ABCB5+ MMIC-dependent tumor growth through promoting RAS protein expression and that cancer therapies targeting HAGE helicase may have broad applications for treating malignant melanoma and potentially other cancer types.

  15. The Helicase HAGE Expressed by Malignant Melanoma-Initiating Cells Is Required for Tumor Cell Proliferation in Vivo*

    PubMed Central

    Linley, Adam J.; Mathieu, Morgan G.; Miles, Amanda K.; Rees, Robert C.; McArdle, Stephanie E. B.; Regad, Tarik

    2012-01-01

    Malignant melanoma-initiating cells (MMIC) are a subpopulation of cells responsible for melanoma tumor growth and progression. They are defined by the expression of the ATP-binding cassette (ABC) subfamily B member 5 (ABCB5). Here, we identified a critical role for the DEAD-box helicase antigen (HAGE) in ABCB5+ MMIC-dependent tumorigenesis and show that HAGE-specific inactivation inhibits melanoma tumor growth mediated by this tumor-initiating population. Knockdown of HAGE led to a significant decrease in RAS protein expression with a concomitant decrease in activation of the AKT and ERK signaling pathways implicated to play an important role in melanoma progression. To confirm that the reduction in NRAS (Neuroblastoma RAS) expression was dependent on the HAGE helicase activity, we showed that NRAS, effectively silenced by siRNA, could be rescued by reintroduction of HAGE in cells lacking HAGE. Furthermore, we provide a mechanism by which HAGE promotes NRAS unwinding in vitro. We also observed using tumor transplantation in Non-obese diabetic/severe combined immunodeficiency mice that the HAGE knockdown in a ABCB5+ melanoma cell line displayed a significant decrease in tumor growth and compared with the control. Our results suggest that the helicase HAGE is required for ABCB5+ MMIC-dependent tumor growth through promoting RAS protein expression and that cancer therapies targeting HAGE helicase may have broad applications for treating malignant melanoma and potentially other cancer types. PMID:22393060

  16. IFN-gamma priming up-regulates IFN-stimulated gene factor 3 (ISGF3) components, augmenting responsiveness of IFN-resistant melanoma cells to type I IFNs.

    PubMed

    Wong, L H; Hatzinisiriou, I; Devenish, R J; Ralph, S J

    1998-06-01

    IFN-stimulated gene factor 3 (ISGF3) mediates transcriptional activation of IFN-sensitive genes (ISGs). The component subunits of ISGF3, STAT1alphabeta, STAT2, and p48-ISGF3gamma, are tyrosine phosphorylated before their assembly into a complex. Subsequently, the ISGF3 complex is translocated to the nucleus. We have recently established that the responsiveness of human melanoma cell lines to type I IFNs correlates directly with their intracellular levels of ISGF3 components, particularly STAT1. In the present study, we show that pretreating IFN-resistant melanoma cell lines with IFN-gamma (IFN-gamma priming) before stimulation with type I IFN also results in increased levels of ISGF3 components and enhanced DNA-binding activation of ISGF3. In addition, IFN-gamma priming of IFN-resistant melanoma cell lines increased expression of type I IFN-induced ISG products, including ISG54, 2'-5'-oligoadenylate synthase, HLA class I, B7-1, and ICAM-1 Ags. Furthermore, IFN-gamma priming enhanced the antiviral effect of IFN-beta on the IFN-resistant melanoma cell line, MM96. These results support a role for IFN-gamma priming in up-regulating ISGF3, thereby augmenting the responsiveness of IFN-resistant melanoma cell lines to type I IFN and providing a molecular basis and justification for using sequential IFN therapy, as proposed by others, to enhance the use of IFNs in the treatment of melanoma.

  17. The Cytolytic Amphipathic β(2,2)-Amino Acid LTX-401 Induces DAMP Release in Melanoma Cells and Causes Complete Regression of B16 Melanoma.

    PubMed

    Eike, Liv-Marie; Mauseth, Brynjar; Camilio, Ketil André; Rekdal, Øystein; Sveinbjørnsson, Baldur

    2016-01-01

    In the present study we examined the ability of the amino acid derivative LTX-401 to induce cell death in cancer cell lines, as well as the capacity to induce regression in a murine melanoma model. Mode of action studies in vitro revealed lytic cell death and release of danger-associated molecular pattern molecules, preceded by massive cytoplasmic vacuolization and compromised lysosomes in treated cells. The use of a murine melanoma model demonstrated that the majority of animals treated with intratumoural injections of LTX-401 showed complete and long-lasting remission. Taken together, these results demonstrate the potential of LTX-401 as an immunotherapeutic agent for the treatment of solid tumors.

  18. Effects of fotemustine or dacarbasine on a melanoma cell line pretreated with therapeutic proton irradiation

    PubMed Central

    Ristić-Fira, Aleksandra M; Korićanac, Lela B; Žakula, Jelena J; Valastro, Lucia M; Iannolo, Gioacchin; Privitera, Giuseppe; Cuttone, Giacomo; Petrović, Ivan M

    2009-01-01

    Background Considering that HTB140 melanoma cells have shown a poor response to either protons or alkylating agents, the effects of a combined use of these agents have been analysed. Methods Cells were irradiated in the middle of the therapeutic 62 MeV proton spread out Bragg peak (SOBP). Irradiation doses were 12 or 16 Gy and are those frequently used in proton therapy. Four days after irradiation cells were treated with fotemustine (FM) or dacarbazine (DTIC). Drug concentrations were 100 and 250 μM, values close to those that produce 50% of growth inhibition. Cell viability, proliferation, survival and cell cycle distribution were assessed 7 days after irradiation that corresponds to more than six doubling times of HTB140 cells. In this way incubation periods providing the best single effects of drugs (3 days) and protons (7 days) coincided at the same time. Results Single proton irradiations have reduced the number of cells to ~50%. FM caused stronger cell inactivation due to its high toxicity, while the effectiveness of DTIC, that was important at short term, almost vanished with the incubation of 7 days. Cellular mechanisms triggered by proton irradiation differently influenced the final effects of combined treatments. Combination of protons and FM did not improve cell inactivation level achieved by single treatments. A low efficiency of the single DTIC treatment was overcome when DTIC was introduced following proton irradiation, giving better inhibitory effects with respect to the single treatments. Most of the analysed cells were in G1/S phase, viable, active and able to replicate DNA. Conclusion The obtained results are the consequence of a high resistance of HTB140 melanoma cells to protons and/or drugs. The inactivation level of the HTB140 human melanoma cells after protons, FM or DTIC treatments was not enhanced by their combined application. PMID:19358719

  19. Effects of fotemustine or dacarbasine on a melanoma cell line pretreated with therapeutic proton irradiation.

    PubMed

    Ristić-Fira, Aleksandra M; Korićanac, Lela B; Zakula, Jelena J; Valastro, Lucia M; Iannolo, Gioacchin; Privitera, Giuseppe; Cuttone, Giacomo; Petrović, Ivan M

    2009-04-09

    Considering that HTB140 melanoma cells have shown a poor response to either protons or alkylating agents, the effects of a combined use of these agents have been analysed. Cells were irradiated in the middle of the therapeutic 62 MeV proton spread out Bragg peak (SOBP). Irradiation doses were 12 or 16 Gy and are those frequently used in proton therapy. Four days after irradiation cells were treated with fotemustine (FM) or dacarbazine (DTIC). Drug concentrations were 100 and 250 microM, values close to those that produce 50% of growth inhibition. Cell viability, proliferation, survival and cell cycle distribution were assessed 7 days after irradiation that corresponds to more than six doubling times of HTB140 cells. In this way incubation periods providing the best single effects of drugs (3 days) and protons (7 days) coincided at the same time. Single proton irradiations have reduced the number of cells to approximately 50%. FM caused stronger cell inactivation due to its high toxicity, while the effectiveness of DTIC, that was important at short term, almost vanished with the incubation of 7 days. Cellular mechanisms triggered by proton irradiation differently influenced the final effects of combined treatments. Combination of protons and FM did not improve cell inactivation level achieved by single treatments. A low efficiency of the single DTIC treatment was overcome when DTIC was introduced following proton irradiation, giving better inhibitory effects with respect to the single treatments. Most of the analysed cells were in G1/S phase, viable, active and able to replicate DNA. The obtained results are the consequence of a high resistance of HTB140 melanoma cells to protons and/or drugs. The inactivation level of the HTB140 human melanoma cells after protons, FM or DTIC treatments was not enhanced by their combined application.

  20. Genetically engineered mesenchymal stromal cells producing TNFα have tumour suppressing effect on human melanoma xenograft.

    PubMed

    Tyciakova, Silvia; Matuskova, Miroslava; Bohovic, Roman; Polakova, Katarina; Toro, Lenka; Skolekova, Svetlana; Kucerova, Lucia

    2015-01-01

    Mesenchymal stromal cells (MSC) are a promising tool for targeted cancer therapy due to their tumour-homing ability. Intrinsic resistance enables the MSC to longer tolerate therapeutic factors, such as prodrug converting enzymes, cytokines and pro-apoptotic proteins. Tumour necrosis factor alpha (TNFα) is known to be cytotoxic to a variety of cancer cells and exert a tumour-destructive capacity. MSC were retrovirally transduced to stable express an exogenous gene encoding the desired therapeutic agent hTNFα. The effect of a TNFα-producing adipose tissue-derived MSC (AT-MSC/hTNFα) was tested on the tumour cell lines of different origins: melanoma (A375), breast carcinoma (SKBR3, MDA-MB-231), colon carcinoma (HT29), ovarian carcinoma (SKOV3) and glioblastoma (U87-MG) cells. The tumour suppressing effect of AT-MSC/hTNFα on A375 melanoma xenografts was monitored in an immunodeficient mouse model in vivo. Engineered AT-MSC are able to constitutively secrete human TNFα protein, induce apoptosis of tumour cell lines via caspase 3/7 activation and inhibit the tumour cell proliferation in vitro. Melanoma A375 and breast carcinoma SKBR3 cells were the most sensitive, and their proliferation in vitro was reduced by conditioned media produced by AT-MSC/hTNFα to 60% and 40%, respectively. The previously reported tumour supportive effect of AT-MSC on subcutaneous A375 melanoma xenograft growth was neutralised and suppressed by engineered AT-MSC stably producing hTNFα. When AT-MSC/hTNFα were coinjected with A375 melanoma cells, the tumour mass inhibition was up to 97.5%. The results of the present study demonstrate that tumour cells respond to hTNFα-based treatment mediated by genetically engineered AT-MSC/hTNFα both in vitro and in vivo. Copyright © 2015 John Wiley & Sons, Ltd.

  1. Wnt/β-catenin signaling inhibitor ICG-001 enhances pigmentation of cultured melanoma cells.

    PubMed

    Kim, Kyung-Il; Jeong, Do-Sun; Jung, Eui Chang; Lee, Jeung-Hoon; Kim, Chang Deok; Yoon, Tae-Jin

    2016-11-01

    Wnt/β-catenin signaling is important in development and differentiation of melanocytes. The object of this study was to evaluate the effects of several Wnt/β-catenin signaling inhibitors on pigmentation using melanoma cells. Melanoma cells were treated with Wnt/β-catenin signaling inhibitors, and then melanin content and tyrosinase activity were checked. Although some inhibitors showed slight inhibition of pigmentation, we failed to observe potential inhibitory effect of those chemicals on pigmentation of HM3KO melanoma cells. Rather, one of powerful Wnt/β-catenin signaling inhibitors, ICG-001, increased the pigmentation of HM3KO melanoma cells. Pigmentation-enhancing effect of ICG-001 was reproducible in other melanoma cell line MNT-1. Consistent with these results. ICG-001 increased the expression of pigmentation-related genes, such as MITF, tyrosinase and TRP1. When ICG-001 was treated, the phosphorylation of CREB was significantly increased. In addition, ICG-001 treatment led to quick increase of intracellular cAMP level, suggesting that ICG-001 activated PKA signaling. The blockage of PKA signaling with pharmaceutical inhibitor H89 inhibited the ICG-001-induced pigmentation significantly. These results suggest that PKA signaling is pivotal in pigmentation process itself, while the importance of Wnt/β-catenin signaling should be emphasized in the context of development and differentiation. Copyright © 2016 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

  2. microRNA-216b inhibits cell proliferation and migration in human melanoma by targeting FOXM1 in vitro and in vivo.

    PubMed

    Sun, Mengyao; Wang, Xiaopeng; Tu, Chen; Wang, Shuang; Qu, Jianqiang; Xiao, Shengxiang

    2017-12-01

    MicroRNAs (miRNAs) play an increasingly important role in cancer growth by coordinately suppressing genes that control cell migration, proliferation, and invasion. The above results can be achieved through the regulation of gene expression by miRNAs by suppressing translation or the direct sequence-specific degradation of the targeted mRNA. In the present study, we indicate that the expression of miR-216b could be effectively repressed both in human melanoma tissues through a comparison with primary melanoma and in human melanoma cell lines through a comparison with a normal human keratinocyte line. Moreover, miR-216b induced a clear decrease in melanoma cell proliferation and migration in vitro. Forkhead box M1 (FOXM1) was confirmed as a target gene of miR-216b, and the overexpression of miR-216b markedly repressed the luciferase activity of reporter plasmids containing the FOXM1 3'-UTR (untranslated region). Furthermore, miR-216b suppressed melanoma cell growth in nude mice in vivo, with the effects of miR-216b overexpression on melanoma cell growth and proliferation reversed by FOXM1 overexpression. The results demonstrated that miR-216b is a tumor suppressor in melanoma, identified the FOXM1 signaling pathway as a target of miR-216b action, and suggested a potential therapeutic role for miR-216b in melanoma. © 2017 International Federation for Cell Biology.

  3. Shifting the Balance of Activating and Inhibitory Natural Killer Receptor Ligands on BRAFV600E Melanoma Lines with Vemurafenib.

    PubMed

    Frazao, Alexandra; Colombo, Marina; Fourmentraux-Neves, Emmanuelle; Messaoudene, Meriem; Rusakiewicz, Sylvie; Zitvogel, Laurence; Vivier, Eric; Vély, Frédéric; Faure, Florence; Dréno, Brigitte; Benlalam, Houssem; Bouquet, Fanny; Savina, Ariel; Pasmant, Eric; Toubert, Antoine; Avril, Marie-Françoise; Caignard, Anne

    2017-07-01

    Over 60% of human melanoma tumors bear a mutation in the BRAF gene. The most frequent mutation is a substitution at codon 600 (V600E), leading to a constitutively active BRAF and overactivation of the MAPK pathway. Patients harboring mutated BRAF respond to kinase inhibitors such as vemurafenib. However, these responses are transient, and relapses are frequent. Melanoma cells are efficiently lysed by activated natural killer (NK) cells. Melanoma cells express several stress-induced ligands that are recognized by activating NK-cell receptors. We have investigated the effect of vemurafenib on the immunogenicity of seven BRAF -mutated melanoma cells to NK cells and on their growth and sensitivity to NK-cell-mediated lysis. We showed that vemurafenib treatment modulated expression of ligands for two activating NK receptors, increasing expression of B7-H6, a ligand for NKp30, and decreasing expression of MICA and ULBP2, ligands for NKG2D. Vemurafenib also increased expression of HLA class I and HLA-E molecules, likely leading to higher engagement of inhibitory receptors (KIRs and NKG2A, respectively), and decreased lysis of vemurafenib-treated melanoma cell lines by cytokine-activated NK cells. Finally, we showed that whereas batimastat (a broad-spectrum matrix metalloprotease inhibitor) increased cell surface ULBP2 by reducing its shedding, vemurafenib lowered soluble ULBP2, indicating that BRAF signal inhibition diminished expression of both cell-surface and soluble forms of NKG2D ligands. Vemurafenib, inhibiting BRAF signaling, shifted the balance of activatory and inhibitory NK ligands on melanoma cells and displayed immunoregulatory effects on NK-cell functional activities. Cancer Immunol Res; 5(7); 582-93. ©2017 AACR . ©2017 American Association for Cancer Research.

  4. The kin17 Protein in Murine Melanoma Cells

    PubMed Central

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

    2015-01-01

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

  5. Identification of DRG-1 As a Melanoma-Associated Antigen Recognized by CD4+ Th1 Cells

    PubMed Central

    Kiniwa, Yukiko; Li, Jiang; Wang, Mingjun; Sun, Chuang; Lee, Jeffrey E.; Wang, Rong-Fu; Wang, Helen Y.

    2015-01-01

    Immunotherapy has emerged as a promising strategy for the treatment of metastatic melanoma. Clinical studies have demonstrated the feasibility of cancer immunotherapy using tumor antigens recognized by CD8+ T cells. However, the overall immune responses induced by these antigens are too weak and transient to induce tumor regression in the majority of patients who received immunization. A growing body of evidence suggests that CD4+ T helper (Th) cells play an important role in antitumor immunity. Therefore, the identification of MHC class II-restricted tumor antigens capable of stimulating CD4+ T cells may provide opportunities for developing effective cancer vaccines. To this end, we describe the identification of developmentally regulated GTP-binding protein 1 (DRG-1) as a melanoma-associated antigen recognized by HLA-DR11-restricted CD4+ Th1 cells. Epitope mapping analysis showed that the DRG1248-268 epitope of DRG-1 was required for T cell recognition. Reverse transcription-polymerase chain reaction revealed that DRG-1 was highly expressed in melanoma cell lines but not in normal tissues. DRG-1 knockdown by lentiviral-based shRNA suppressed melanoma cell proliferation and soft agar colony formation. Taken together, these data suggest that DRG-1 plays an important role in melanoma cell growth and transformation, indicating that DRG1 may represent a novel target for CD4+ T cell-mediated immunotherapy in melanoma. PMID:25993655

  6. Identification of DRG-1 As a Melanoma-Associated Antigen Recognized by CD4+ Th1 Cells.

    PubMed

    Kiniwa, Yukiko; Li, Jiang; Wang, Mingjun; Sun, Chuang; Lee, Jeffrey E; Wang, Rong-Fu; Wang, Helen Y

    2015-01-01

    Immunotherapy has emerged as a promising strategy for the treatment of metastatic melanoma. Clinical studies have demonstrated the feasibility of cancer immunotherapy using tumor antigens recognized by CD8(+) T cells. However, the overall immune responses induced by these antigens are too weak and transient to induce tumor regression in the majority of patients who received immunization. A growing body of evidence suggests that CD4(+) T helper (Th) cells play an important role in antitumor immunity. Therefore, the identification of MHC class II-restricted tumor antigens capable of stimulating CD4(+) T cells may provide opportunities for developing effective cancer vaccines. To this end, we describe the identification of developmentally regulated GTP-binding protein 1 (DRG-1) as a melanoma-associated antigen recognized by HLA-DR11-restricted CD4(+) Th1 cells. Epitope mapping analysis showed that the DRG1248-268 epitope of DRG-1 was required for T cell recognition. Reverse transcription-polymerase chain reaction revealed that DRG-1 was highly expressed in melanoma cell lines but not in normal tissues. DRG-1 knockdown by lentiviral-based shRNA suppressed melanoma cell proliferation and soft agar colony formation. Taken together, these data suggest that DRG-1 plays an important role in melanoma cell growth and transformation, indicating that DRG1 may represent a novel target for CD4(+) T cell-mediated immunotherapy in melanoma.

  7. Effects of Malignant Melanoma Initiating Cells on T-Cell Activation

    PubMed Central

    Schatton, Tobias; Schütte, Ute; Frank, Markus H.

    2016-01-01

    Although human malignant melanoma is a highly immunogenic cancer, both the endogenous antitumor immune response and melanoma immunotherapy often fail to control neoplastic progression. Accordingly, characterizing melanoma cell subsets capable of evading antitumor immunity could unravel optimized treatment strategies that might reduce morbidity and mortality from melanoma. By virtue of their preferential capacity to modulate antitumor immune responses and drive inexorable tumor growth and progression, malignant melanoma-initiating cells (MMICs) warrant closer investigation to further elucidate the cellular and molecular mechanisms underlying melanoma immune evasion and immunotherapy resistance. Here we describe methodologies that enable the characterization of immunoregulatory effects of purified MMICs versus melanoma bulk populations in coculture with syngeneic or allogeneic lymphocytes, using [3H] thymidine incorporation, enzyme-linked immunosorbent spot (ELISPOT), or ELISA assays. These assays were traditionally developed to analyze alloimmune processes and we successfully adapted them for the study of tumor-mediated immunomodulatory functions. PMID:26786883

  8. Efficient isolation of human parainfluenza viruses 1 and 3 using MNT-1, a human malignant melanoma cell line system that exhibits an apparent cytopathic effect.

    PubMed

    Sato, Ko; Watanabe, Oshi; Ohmiya, Suguru; Chiba, Fumiko; Hayashi, Masahiro; Suzuki, Tamio; Kawakami, Kazuyoshi; Nishimura, Hidekazu

    2016-11-01

    Isolation of human parainfluenza virus (HPIV) serotypes 1 and 3 from clinical specimens is not very efficient because of the lack of a cell culture system capable of inducing CPE. In this study, the utility of a melanoma cell line, MNT-1, that allows HPIV growth and displays CPE was demonstrated. In particularly, the efficiency of isolating HPIV1 and HPIV3 using MNT-1 was greater than for cell lines conventionally used for HPIV isolation. Our demonstrated efficacy of HPIV1 and HPIV3 isolation with apparent CPE using the MNT-1 cell culture system has the potential to improve virus isolation from clinical specimens. © 2016 The Societies and John Wiley & Sons Australia, Ltd.

  9. 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.

    PubMed

    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.

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

    PubMed

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

    2017-08-01

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

  11. VEGFR-1 Expressed by Malignant Melanoma-Initiating Cells Is Required for Tumor Growth

    PubMed Central

    Frank, Natasha Y.; Schatton, Tobias; Kim, Soo; Zhan, Qian; Wilson, Brian J.; Ma, Jie; Saab, Karim R.; Osherov, Veronika; Widlund, Hans R.; Gasser, Martin; Waaga-Gasser, Ana-Maria; Kupper, Thomas S.; Murphy, George F.; Frank, Markus H.

    2011-01-01

    Melanoma growth is driven by malignant melanoma-initiating cells (MMIC) identified by expression of the ATP-binding cassette (ABC) member ABCB5. ABCB5+ melanoma subpopulations have been shown to overexpress the vasculogenic differentiation markers CD144 (VE-cadherin) and TIE1 and are associated with CD31− vasculogenic mimicry (VM), an established biomarker associated with increased patient mortality. Here we identify a critical role for VEGFR-1 signaling in ABCB5+ MMIC-dependent VM and tumor growth. Global gene expression analyses, validated by mRNA and protein determinations, revealed preferential expression of VEGFR-1 on ABCB5+ tumor cells purified from clinical melanomas and established melanoma lines. In vitro, VEGF induced the expression of CD144 in ABCB5+ subpopulations that constitutively expressed VEGFR-1 but not in ABCB5− bulk populations that were predominantly VEGFR-1−. In vivo, melanoma-specific shRNA-mediated knockdown of VEGFR-1 blocked the development of ABCB5+ VM morphology and inhibited ABCB5+ VM-associated production of the secreted melanoma mitogen laminin. Moreover, melanoma-specific VEGFR-1 knockdown markedly inhibited tumor growth (by >90%). Our results show that VEGFR-1 function in MMIC regulates VM and associated laminin production and show that this function represents one mechanism through which MMICs promote tumor growth. PMID:21212411

  12. Melanoma cell-intrinsic PD-1 receptor functions promote tumor growth

    PubMed Central

    Kleffel, Sonja; Posch, Christian; Barthel, Steven R.; Mueller, Hansgeorg; Schlapbach, Christoph; Guenova, Emmanuella; Elco, Christopher P.; Lee, Nayoung; Juneja, Vikram R.; Zhan, Qian; Lian, Christine G.; Thomi, Rahel; Hoetzenecker, Wolfram; Cozzio, Antonio; Dummer, Reinhard; Mihm, Martin C.; Flaherty, Keith T.; Frank, Markus H.; Murphy, George F.; Sharpe, Arlene H.; Kupper, Thomas S.; Schatton, Tobias

    2015-01-01

    SUMMARY Therapeutic antibodies targeting programmed cell death-1 (PD-1) activate tumor-specific immunity and have shown remarkable efficacy in the treatment of melanoma. Yet, little is known about tumor cell-intrinsic PD-1 pathway effects. Here we show that murine and human melanomas contain PD-1-expressing cancer subpopulations and demonstrate that melanoma cell-intrinsic PD-1 promotes tumorigenesis, even in mice lacking adaptive immunity. PD-1 inhibition on melanoma cells by RNA interference, blocking antibodies, or mutagenesis of melanoma-PD-1 signaling motifs suppresses tumor growth in immunocompetent, immunocompromised and PD-1-deficient tumor graft recipient mice. Conversely, melanoma-specific PD-1 overexpression enhances tumorigenicity, as does engagement of melanoma-PD-1 by its ligand, PD-L1, whereas melanoma-PD-L1 inhibition or knockout of host-PD-L1 attenuate growth of PD-1-positive melanomas. Mechanistically, the melanoma-PD-1 receptor modulates downstream effectors of mTOR signaling. Our results identify melanoma cell-intrinsic functions of the PD-1:PD-L1 axis in tumor growth and suggest that blocking melanoma-PD-1 might contribute to the striking clinical efficacy of anti-PD-1 therapy. PMID:26359984

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

    PubMed Central

    2013-01-01

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

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

    PubMed

    Niero, Evandro Luís de Oliveira; Machado-Santelli, Gláucia Maria

    2013-05-23

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

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

    PubMed

    Hata, Keishi; Hori, Kazuyuki; Takahashi, Saori

    2002-05-01

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

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

    PubMed

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

    2016-08-09

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

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

    PubMed Central

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

    2016-01-01

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

  18. The Cytolytic Amphipathic β(2,2)-Amino Acid LTX-401 Induces DAMP Release in Melanoma Cells and Causes Complete Regression of B16 Melanoma

    PubMed Central

    Eike, Liv-Marie; Mauseth, Brynjar; Camilio, Ketil André; Rekdal, Øystein; Sveinbjørnsson, Baldur

    2016-01-01

    In the present study we examined the ability of the amino acid derivative LTX-401 to induce cell death in cancer cell lines, as well as the capacity to induce regression in a murine melanoma model. Mode of action studies in vitro revealed lytic cell death and release of danger-associated molecular pattern molecules, preceded by massive cytoplasmic vacuolization and compromised lysosomes in treated cells. The use of a murine melanoma model demonstrated that the majority of animals treated with intratumoural injections of LTX-401 showed complete and long-lasting remission. Taken together, these results demonstrate the potential of LTX-401 as an immunotherapeutic agent for the treatment of solid tumors. PMID:26881822

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

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

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

    2014-08-08

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

  20. GSK-3beta inhibition enhances sorafenib-induced apoptosis in melanoma cell lines.

    PubMed

    Panka, David J; Cho, Daniel C; Atkins, Michael B; Mier, James W

    2008-01-11

    Glycogen synthase kinase-3beta (GSK-3beta) can participate in the induction of apoptosis or, alternatively, provide a survival signal that minimizes cellular injury. We previously demonstrated that the multikinase inhibitor sorafenib induces apoptosis in melanoma cell lines. In this report, we show that sorafenib activates GSK-3beta in multiple subcellular compartments and that this activation undermines the lethality of the drug. Pharmacologic inhibition and/or down-modulation of the kinase enhances sorafenib-induced apoptosis as determined by propidium iodide staining and by assessing the mitochondrial release of apoptosis-inducing factor and Smac/DIABLO. Conversely, the forced expression of a constitutively active form of the enzyme (GSK-3beta(S9A)) protects the cells from the apoptotic effects of the drug. This protective effect is associated with a marked increase in basal levels of Bcl-2, Bcl-x(L), and survivin and a diminution in the degree to which these anti-apoptotic proteins are down-modulated by sorafenib exposure. Sorafenib down-modulates the pro-apoptotic Bcl-2 family member Noxa in cells with high constitutive GSK-3beta activity. Pharmacologic inhibition of GSK-3beta prevents the disappearance of Noxa induced by sorafenib and enhances the down-modulation of Mcl-1. Down-modulation of Noxa largely eliminates the enhancing effect of GSK-3 inhibition on sorafenib-induced apoptosis. These data provide a strong rationale for the use of GSK-3beta inhibitors as adjuncts to sorafenib treatment and suggest that preservation of Noxa may contribute to their efficacy.

  1. Proteomic Analysis of Laser Microdissected Melanoma Cells from Skin Organ Cultures

    PubMed Central

    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

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

    PubMed

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

    2015-11-01

    Cutaneous malignant melanoma is the leading cause of death from skin diseases and is often associated with activating mutations of the proto-oncogene BRAF. To develop more effective strategies for the prevention or treatment of melanoma, we have examined the inhibitory effects of silymarin, a flavanoid from Silybum marianum, on melanoma cells. Using A375 (BRAF-mutated) and Hs294t (non BRAF-mutated but highly metastatic) human melanoma cell lines, we found that in vitro treatment with silymarin resulted in a dose-dependent: (i) reduction in cell viability; (ii) enhancement of either Go/G1 (A375) or G2-M (Hs294t) phase cell cycle arrest with corresponding alterations in cyclins and cyclin-dependent kinases; and (iii) induction of apoptosis. The silymarin-induced apoptosis of human melanoma cells was associated with a reduction in the levels of anti-apoptotic proteins (Bcl-2 and Bcl-xl), an increase in the levels of pro-apoptotic protein (Bax), and activation of caspases. Further, oral administration of silymarin (500 mg/kg body weight/2× a week) significantly inhibited (60%, P < 0.01) the growth of BRAF-mutated A375 melanoma tumor xenografts, and this was associated with: (i) inhibition of cell proliferation; (ii) induction of apoptosis of tumor cells; (iii) alterations in cell cycle regulatory proteins; and (iv) reduced expression of tumor angiogenic biomarkers in tumor xenograft tissues. These results indicate that silymarin may have a chemotherapeutic effect on human melanoma cell growth and warrant its further evaluation. © 2014 Wiley Periodicals, Inc.

  3. A high molecular weight-melanoma associated antigen-specific chimeric antigen receptor redirects lymphocytes to target human melanomas

    PubMed Central

    Burns, William R.; Zhao, Yangbing; Frankel, Timothy L.; Hinrichs, Christian S.; Zheng, Zhili; Xu, Hui; Feldman, Steven A.; Ferrone, Soldano; Rosenberg, Steven A.; Morgan, Richard A.

    2011-01-01

    Immunotherapy, particularly the adoptive cell transfer (ACT) of tumor infiltrating lymphocytes (TIL), is a very promising therapy for metastatic melanoma. Some patients unable to receive TIL have been successfully treated with autologous peripheral blood lymphocytes (PBL), genetically modified to express HLA class I antigen restricted, melanoma antigen-reactive T-cell receptors; however, substantial numbers of patients remain ineligible due to the lack of expression of the restricting HLA class I allele. We sought to overcome this limitation by designing a non-MHC-restricted, chimeric antigen receptor (CAR) targeting the high molecular weight-melanoma associated antigen (HMW-MAA), which is highly expressed on over 90% of human melanomas but has a restricted distribution in normal tissues. HMW-MAA-specific CARs containing an antigen recognition domain based on variations of the HMW-MAA-specific monoclonal antibody (mAb) 225.28S and a T-cell activation domain based on combinations of CD28, 4-1BB, and CD3ζ activation motifs were constructed within a retroviral vector to allow stable gene transfer into cells and their progeny. Following optimization of the HMW-MAA-specific CAR for expression and function in human PBL, these gene-modified T cells secreted cytokines, were cytolytic, and proliferated in response to HMW-MAA expressing cell lines. Furthermore, the receptor functioned in both CD4+ and CD8+ cells, was non-MHC-restricted, and reacted against explanted human melanomas. To evaluate this HMW-MAA-specific CAR in patients with metastatic melanoma, we developed a clinical-grade retroviral packaging line. This may represent a novel means to treat the majority of patients with advanced melanoma, most notably those unable to receive current ACT therapies. PMID:20395199

  4. Piceatannol induced apoptosis through up-regulation of microRNA-181a in melanoma cells.

    PubMed

    Du, Maotao; Zhang, Zhong; Gao, Tao

    2017-10-17

    Melanoma took top position among the lethal cancers and, despite there have been some great attempts made to increase the natural life of patients with metastatic disease, long-lasting and complete remissions are few. Piceatannol, owns the similar function as resveratrol, has been defined as an anti-cancer agent playing important role in inhibition of proliferation, migration and metastasis in various cancer. Thus, we aim to investigate the anti-cancer effect and mechanisms of piceatannol in melanoma cells. Melanoma cell lines WM266-4 and A2058 were treated either with or without piceatannol. Cell viability and cell apoptosis were assessed by using MTT and Annexin V/PI assay, respectively. Cells were transfected with specific miRNA using Lipfectamine 2000. miRNA bingding ability to 3'-UTR region within specific gene was assed by firefly luciferase analysis. Gene and protein expression was eveluated by qRT-PCR and western blot analysis, respectively. Our study showed that piceatannol inhibited WM266-4 and A2058 cells growth and induced apoptosis. Totally, 16 differentially expressed miRNAs were screened out including 8 up-regulated and 8 down-regulated miRNAs. Expression level of miR-181a is significantly higher in piceatannol-treated cells than normal control and is lower in melanoma cancer tissues than its adjacent normal tissues. Bcl-2 is a target gene of miR-181a. Moreover, silencing of miR-181a reverses the decrease of cell viability induced by piceatannol in WM266-4 and A2058 cells. Taken together, present study uncovered the ability of piceatannol to repress melanoma cell growth and clarified the contribution of miR-181a in the anticancer role of piceatannol. The present study proposes that piceatannol can be taken into account to be a hopeful anticancer agent for melanoma.

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

    PubMed

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

    2011-06-01

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

  6. Vemurafenib potently induces endoplasmic reticulum stress-mediated apoptosis in BRAFV600E melanoma cells

    PubMed Central

    Beck, Daniela; Niessner, Heike; Smalley, Keiran S.M.; Flaherty, Keith; Paraiso, Kim H.T.; Busch, Christian; Sinnberg, Tobias; Vasseur, Sophie; Iovanna, Juan Lucio; Drießen, Stefan; Stork, Björn; Wesselborg, Sebastian; Schaller, Martin; Biedermann, Tilo; Bauer, Jürgen; Lasithiotakis, Konstantinos; Weide, Benjamin; Eberle, Jürgen; Schittek, Birgit; Schadendorf, Dirk; Garbe, Claus; Kulms, Dagmar; Meier, Friedegund

    2013-01-01

    The V600E mutation in the kinase BRAF is frequently detected in melanomas and results in constitutive activation of BRAF, which then promotes cell proliferation by the mitogen-activated protein kinase (MAPK) signaling pathway. Although the BRAFV600E kinase inhibitor vemurafenib has remarkable antitumor activity in patients with BRAFV600E-mutated melanoma, its effects are limited by the onset of drug resistance. We found that exposure of melanoma cell lines with the BRAFV600E mutation to vemurafenib decreased the abundance of anti-apoptotic proteins and induced intrinsic mitochondrial apoptosis. Vemurafenib-treated melanoma cells showed increased cytosolic concentration of calcium, a potential trigger for endoplasmic reticulum (ER) stress, which can lead to apoptosis. Consistent with an ER stress-induced response, vemurafenib decreased the abundance of the ER chaperone protein GRP78, increased the abundance of the spliced isoform of the transcription factor X-box protein 1 (XBP1) (which transcriptionally activates genes involved in ER stress responses), increased the phosphorylation of the translation initiation factor eIF2α (which would be expected to inhibit protein synthesis), and induced the expression of ER stress-related genes. Knockdown of the ER stress response protein ATF4 significantly reduced vemurafenib-induced apoptosis. Moreover, the ER stress inducer thapsigargin prevented invasive growth of tumors formed from vemurafenib-sensitive melanoma cells in vivo. In melanoma cells with low sensitivity or resistance to vemurafenib, combination treatment with thapsigargin augmented or induced apoptosis. Thus, thapsigargin or other inducers of ER stress may be useful in combination therapies to overcome vemurafenib resistance. PMID:23362240

  7. A Novel Inhibitor of STAT3 Activation Is Efficacious Against Established Central Nervous System Melanoma and Inhibits Regulatory T Cells

    PubMed Central

    Kong, Ling-Yuan; Abou-Ghazal, Mohamed K.; Wei, Jun; Chakraborty, Arup; Sun, Wei; Qiao, Wei; Fuller, Gregory N.; Fokt, Izabela; Grimm, Elizabeth A.; Schmittling, Robert J.; Archer, Gary E.; Sampson, John H.; Priebe, Waldemar; Heimberger, Amy B.

    2008-01-01

    Purpose Activation of STAT3 has been identified as a central mediator of melanoma growth and metastasis. We hypothesized that WP1066, a novel STAT3 blockade agent, has marked antitumor activity, even against the melanoma metastasis to brain, a site typically refractory to therapies. Experimental Design The antitumor activities and related mechanisms of WP1066 were investigated both in vitro on melanoma cell lines and in vivo on mice with subcutaneously syngeneic melanoma or with intracerebral melanoma tumors. Results WP1066 achieved an IC50 of 1.6 μM, 2.3 μM, and 1.5 μM against melanoma cell line A375, B16 and B16EGFRvIII, respectively. WP1066 suppressed the phosphorylation of JAK2 and STAT3 (Tyr705) in these cells. Tumor growth in mice with subcutaneously established syngeneic melanoma was markedly inhibited by WP1066 compared with that in controls. Long-term survival (>78 days) was observed in 80% of mice with established intracerebral syngeneic melanoma treated with 40 mg/kg of WP1066 in contrast to control mice who survived for a median of 15 days. Although WP1066 did not induce immunological memory or enhance humoral responses to EGFRvIII, this compound reduced the production of immunosuppressive cytokines and chemokines (TGF-β, RANTES, MCP-1, VEGF), markedly inhibited natural and inducible Treg proliferation, and significantly increased cytotoxic immune responses of T cells. Conclusions The antitumor cytotoxic effects of WP1066 and its ability to induce antitumor immune responses suggest that this compound has potential for the effective treatment of melanoma metastatic to brain. PMID:18794085

  8. Iterative sorting reveals CD133+ and CD133- melanoma cells as phenotypically distinct populations.

    PubMed

    Grasso, Carole; Anaka, Matthew; Hofmann, Oliver; Sompallae, Ramakrishna; Broadley, Kate; Hide, Winston; Berridge, Michael V; Cebon, Jonathan; Behren, Andreas; McConnell, Melanie J

    2016-09-09

    The heterogeneity and tumourigenicity of metastatic melanoma is attributed to a cancer stem cell model, with CD133 considered to be a cancer stem cell marker in melanoma as well as other tumours, but its role has remained controversial. We iteratively sorted CD133+ and CD133- cells from 3 metastatic melanoma cell lines, and observed tumourigenicity and phenotypic characteristics over 7 generations of serial xeno-transplantation in NOD/SCID mice. We demonstrate that iterative sorting is required to make highly pure populations of CD133+ and CD133- cells from metastatic melanoma, and that these two populations have distinct characteristics not related to the cancer stem cell phenotype. In vitro, gene set enrichment analysis indicated CD133+ cells were related to a proliferative phenotype, whereas CD133- cells were of an invasive phenotype. However, in vivo, serial transplantation of CD133+ and CD133- tumours over 7 generations showed that both populations were equally able to initiate and propagate tumours. Despite this, both populations remained phenotypically distinct, with CD133- cells only able to express CD133 in vivo and not in vitro. Loss of CD133 from the surface of a CD133+ cell was observed in vitro and in vivo, however CD133- cells derived from CD133+ retained the CD133+ phenotype, even in the presence of signals from the tumour microenvironment. We show for the first time the necessity of iterative sorting to isolate pure marker-positive and marker-negative populations for comparative studies, and present evidence that despite CD133+ and CD133- cells being equally tumourigenic, they display distinct phenotypic differences, suggesting CD133 may define a distinct lineage in melanoma.

  9. Differential PAX3 functions in normal skin melanocytes and melanoma cells

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

    Medic, Sandra; Rizos, Helen; Ziman, Mel, E-mail: m.ziman@ecu.edu.au

    2011-08-12

    Highlights: {yields} PAX3 retains embryonic roles in adult melanocytes and melanoma cells. {yields} Promotes 'stem' cell-like phenotype via NES and SOX9 in both cells types. {yields} Regulates melanoma and melanocyte migration through MCAM and CSPG4. {yields} PAX3 regulates melanoma but not melanocyte proliferation via TPD52. {yields} Regulates melanoma cell (but not melanocyte) survival via BCL2L1 and PTEN. -- Abstract: The PAX3 transcription factor is the key regulator of melanocyte development during embryogenesis and is also frequently found in melanoma cells. While PAX3 is known to regulate melanocyte differentiation, survival, proliferation and migration during development, it is not clear if itsmore » function is maintained in adult melanocytes and melanoma cells. To clarify this we have assessed which genes are targeted by PAX3 in these cells. We show here that similar to its roles in development, PAX3 regulates complex differentiation networks in both melanoma cells and melanocytes, in order to maintain cells as 'stem' cell-like (via NES and SOX9). We show also that mediators of migration (MCAM and CSPG4) are common to both cell types but more so in melanoma cells. By contrast, PAX3-mediated regulation of melanoma cell proliferation (through TPD52) and survival (via BCL2L1 and PTEN) differs from that in melanocytes. These results suggest that by controlling cell proliferation, survival and migration as well as maintaining a less differentiated 'stem' cell like phenotype, PAX3 may contribute to melanoma development and progression.« less

  10. The antiproliferative effect of coumarins on several cancer cell lines.

    PubMed

    Kawaii, S; Tomono, Y; Ogawa, K; Sugiura, M; Yano, M; Yoshizawa, Y

    2001-01-01

    Twenty-one coumarins were examined for their antiproliferative activity towards several cancer cell lines, namely lung carcinoma (A549), melanin pigment producing mouse melanoma (B16 melanoma 4A5), human T-cell leukemia (CCRF-HSB-2), and human gastric cancer, lymph node metastasized (TGBC11TKB). The structure-activity relationship established from the results revealed that the 6,7-dihydroxy moiety had an important role for their antiproliferative activity. Analysis of cell cycle distribution indicated that esculetin-treated cells accumulated in the G1 (at 400 microM) or in S phase (at 100 microM).

  11. Sub-apoptotic dosages of pro-oxidant vitamin cocktails sensitize human melanoma cells to NK cell lysis.

    PubMed

    Tremante, Elisa; Santarelli, Lory; Lo Monaco, Elisa; Sampaoli, Camilla; Ingegnere, Tiziano; Guerrieri, Roberto; Tomasetti, Marco; Giacomini, Patrizio

    2015-10-13

    Alpha-tocopheryl succinate (αTOS), vitamin K3 (VK3) and vitamin C (ascorbic acid, AA) were previously shown to synergistically promote different death pathways in carcinoma cells, depending on their concentrations and combinations. Similar effects were observed herein in melanoma cells, although αTOS behaved as an antagonist. Interestingly, suboptimal cell death-inducing concentrations (1.5 μM αTOS/20 μM AA/0.2 μM VK3) effectively up-regulated activating Natural Killer (NK) cell ligands, including MICA (the stress-signaling ligand of the NKG2D receptor), and/or the ligands of at least one of the natural cytotoxicity receptors (NKp30, NKp44 and NKp46) in 5/6 melanoma cell lines. Only an isolated MICA down-regulation was seen. HLA class I, HLA class II, ULBP1, ULBP2, ULBP3, Nectin-2, and PVR displayed little, if any, change in expression. Ligand up-regulation resulted in improved lysis by polyclonal NK cells armed with the corresponding activating receptors. These results provide the first evidence for concerted induction of cell death by cell-autonomous and extrinsic (immune) mechanisms. Alarming the immune system much below the cell damage threshold may have evolved as a sensitive readout of neoplastic transformation and oxidative stress. Cocktails of vitamin analogues at slightly supra-physiological dosages may find application as mild complements of melanoma treatment, and in chemoprevention.

  12. Sub-apoptotic dosages of pro-oxidant vitamin cocktails sensitize human melanoma cells to NK cell lysis

    PubMed Central

    Tremante, Elisa; Santarelli, Lory; Monaco, Elisa Lo; Sampaoli, Camilla; Ingegnere, Tiziano; Guerrieri, Roberto

    2015-01-01

    Alpha-tochopheryl succinate (αTOS), vitamin K3 (VK3) and vitamin C (ascorbic acid, AA) were previously shown to synergistically promote different death pathways in carcinoma cells, depending on their concentrations and combinations. Similar effects were observed herein in melanoma cells, although αTOS behaved as an antagonist. Interestingly, suboptimal cell death-inducing concentrations (1.5 μM αTOS/20 μM AA/0.2 μM VK3) effectively up-regulated activating Natural Killer (NK) cell ligands, including MICA (the stress-signaling ligand of the NKG2D receptor), and/or the ligands of at least one of the natural cytotoxicity receptors (NKp30, NKp44 and NKp46) in 5/6 melanoma cell lines. Only an isolated MICA down-regulation was seen. HLA class I, HLA class II, ULBP1, ULBP2, ULBP3, Nectin-2, and PVR displayed little, if any, change in expression. Ligand up-regulation resulted in improved lysis by polyclonal NK cells armed with the corresponding activating receptors. These results provide the first evidence for concerted induction of cell death by cell-autonomous and extrinsic (immune) mechanisms. Alarming the immune system much below the cell damage threshold may have evolved as a sensitive readout of neoplastic transformation and oxidative stress. Cocktails of vitamin analogues at slightly supra-physiological dosages may find application as mild complements of melanoma treatment, and in chemoprevention. PMID:26427039

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

    PubMed

    Glud, Martin; Manfé, Valentina; Biskup, Edyta; Holst, Line; Dirksen, Anne Marie Ahlburg; Hastrup, Nina; Nielsen, Finn C; Drzewiecki, Krzysztof T; Gniadecki, Robert

    2011-06-01

    MicroRNAs (miRNAs) are small noncoding RNA molecules involved in gene regulation. Aberrant expression of miRNA has been associated with the development or progression of several diseases, including cancer. In a previous study, we found that the expression of miRNA-125b (miR-125b) was two-fold lower in malignant melanoma producing lymph node micrometastases than in nonmetastasizing tumors. To get further insight into the functional role of miR-125b, we assessed whether its overexpression or silencing affects apoptosis, proliferation, or senescence in melanoma cell lines. We showed that overexpression of miR-125b induced typical senescent cell morphology, including increased cytoplasmatic/nucleus ratio and intensive cytoplasmatic β-galactosidase expression. In contrast, inhibition of miR-125b resulted in 30-35% decreased levels of spontaneous apoptosis. We propose that downregulation of miR-125b in an early cutaneous malignant melanoma can contribute to the increased metastatic capability of this tumor.

  14. Ultraviolet mutagenesis in a plasmid vector replicated in lymphoid cells from patient with the melanoma-prone disorder dysplastic nevus syndrome

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

    Seetharam, S.; Waters, H.L.; Seidman, M.M.

    The hereditary dysplastic nevus syndrome (DNS) is an autosomal dominant disorder in which affected individuals have increased numbers of dysplastic (premalignant) nevi and a greater than 100-fold increased risk of developing cutaneous melanoma. Epstein-Barr virus-transformed lymphoblastoid cell lines from patients with hereditary DNS have been shown to be hypermutable to UV radiation. To examine the mechanism involved in this UV hypermutability, we used a shuttle vector plasmid, pZ189, which carries a 160-base pair marker gene, supF, and can replicate in human cells. pZ189 was treated with UV radiation and transfected into DNS6BE, a lymphoblastoid cell line from a patient withmore » hereditary DNS. Plasmid survival after UV was similar with the DNS6BE line and with a lymphoblastoid cell line from a normal donor. Plasmid mutation frequency was greater with the DNS line in accord with the DNS cellular hypermutability. Base sequence analysis was performed on 69 mutated plasmids recovered from the DNS line. There were significantly more plasmids with single base substitution mutations (P less than 0.01) in comparison to UV-treated plasmids passed through normal fibroblasts. pZ189 hypermutability and an increased frequency of single base substitutions was previously found with a cell line from a melanoma-prone xeroderma pigmentosum patient. These differences may be related to the increased melanoma susceptibility in both DNS and xeroderma pigmentosum.« less

  15. The proliferation of malignant melanoma cells could be inhibited by ranibizumab via antagonizing VEGF through VEGFR1.

    PubMed

    Li, Jiao; Cui, Yan; Wang, Qin; Guo, Dadong; Pan, Xuemei; Wang, Xingrong; Bi, Hongsheng; Chen, Wei; Liu, Zhengfeng; Zhao, Shengya

    2014-01-01

    Angiogenesis is an important mediator in tumor progression. Vascular endothelial growth factor (VEGF) is one of the major cytokines that can influence angiogenesis. However, the potential mechanism of tumor growth inhibition through anti-VEGF agents is still unclear. This study was performed to examine whether ranibizumab could inhibit malignant melanoma growth in vitro and to determine the safety of ranibizumab on human adult retinal pigment epithelium cell line (ARPE-19 cells). Malignant melanoma cells obtained from a clinic were cultured in vitro. VEGF concentrations secreted by malignant melanoma cells and the ARPE-19 cells were examined by enzyme-linked immunosorbent assay (ELISA). The two kinds of cells were both treated with VEGF and its antagonist, ranibizumab. The dynamic changes of the two types of cells were monitored by real-time cell electronic sensing (RT-CES) assay. The effect of ranibizumab on both types of cells was verified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl (MTT) assay. The expression of VEGF receptor 1 (VEGFR1) RNA in uveal melanoma was further investigated through the PCR technique. The levels of VEGF secreted by malignant melanoma cells were much higher than those of ARPE-19 cells, and were markedly decreased in the action of 0.1 mg/ml ranibizumab. However, there was no obvious reduction of VEGF in the presence of ranibizumab for ARPE-19 (p>0.05). Meanwhile, RT-CES showed that the viability of malignant melanoma cells increased greatly in the presence of VEGF. When VEGF was 20 ng/ml, viability of the malignant melanoma cells increased by 40% compared with the negative control. There was no evident effect on proliferation of ARPE-19 (p>0.05). Furthermore, the growth of malignant melanoma cells was obviously inhibited after ranibizumab intervention. When ranibizumab was administered at 0.25 mg/ml, the survival rate of the malignant melanoma cells decreased to 57.5%. Nevertheless, low-dose exposure to ranibizumab had only a slight

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

    PubMed

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

    2013-08-14

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

  17. THAP5 is a DNA-binding transcriptional repressor that is regulated in melanoma cells during DNA damage-induced cell death

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

    Balakrishnan, Meenakshi P.; Cilenti, Lucia; Ambivero, Camilla

    2011-01-07

    Research highlights: {yields} THAP5 is a DNA-binding protein and a transcriptional repressor. {yields} THAP5 is induced in melanoma cells upon exposure to UV or treatment with cisplatin. {yields} THAP5 induction correlates with the degree of apoptosis in melanoma cell population. {yields} THAP5 is a pro-apoptotic protein involved in melanoma cell death. -- Abstract: THAP5 was originally isolated as a specific interactor and substrate of the mitochondrial pro-apoptotic Omi/HtrA2 protease. It is a human zinc finger protein characterized by a restricted pattern of expression and the lack of orthologs in mouse and rat. The biological function of THAP5 is unknown butmore » our previous studies suggest it could regulate G2/M transition in kidney cells and could be involved in human cardiomyocyte cell death associated with coronary artery disease (CAD). In this report, we expanded our studies on the properties and function of THAP5 in human melanoma cells. THAP5 was expressed in primary human melanocytes as well as in all melanoma cell lines that were tested. THAP5 protein level was significantly induced by UV irradiation or cisplatin treatment, conditions known to cause DNA damage. The induction of THAP5 correlated with a significant increase in apoptotic cell death. In addition, we show that THAP5 is a nuclear protein that could recognize and bind a specific DNA motif. THAP5 could also repress the transcription of a reporter gene in a heterologous system. Our work suggests that THAP5 is a DNA-binding protein and a transcriptional repressor. Furthermore, THAP5 has a pro-apoptotic function and it was induced in melanoma cells under conditions that promoted cell death.« less

  18. An electrochemical immunosensing method for detecting melanoma cells.

    PubMed

    Seenivasan, Rajesh; Maddodi, Nityanand; Setaluri, Vijaysaradhi; Gunasekaran, Sundaram

    2015-06-15

    An electrochemical immunosensing method was developed to detect melanoma cells based on the affinity between cell surface melanocortin 1 receptor (MC1R) antigen and anti-MC1R antibody (MC1R-Ab). The MC1R-Abs were immobilized in amino-functionalized silica nanoparticles (n-SiNPs)-polypyrrole (PPy) nanocomposite modified on working electrode surface of screen-printed electrode (SPE). Cyclic voltammetry was employed, with the help of redox mediator ([Fe(CN)6](3-)), to measure the change in anodic oxidation peak current arising due to the specific interaction between MC1R antigens and MC1R-Abs when the target melanoma cells are present in the sample. Various factors affecting the sensor performance, such as the amount of MC1R-Abs loaded, incubation time with the target melanoma cells, the presence of interfering non-melanoma cells, were tested and optimized over different expected melanoma cell loads in the range of 50-7500 cells/2.5 mL. The immunosensor is highly sensitive (20 cells/mL), specific, and reproducible, and the antibody-loaded electrode in ready-to-use stage is stable over two weeks. Thus, in conjunction with a microfluidic lab-on-a-chip device our electrochemical immunosensing approach may be suitable for highly sensitive, selective, and rapid detection of circulating tumor cells (CTCs) in blood samples. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. p54nrb is a new regulator of progression of malignant melanoma.

    PubMed

    Schiffner, Susanne; Zimara, Nicole; Schmid, Rainer; Bosserhoff, Anja-Katrin

    2011-08-01

    Nuclear RNA-binding protein p54(nrb) and its murine homolog NonO are known to be involved in a variety of nuclear processes including transcription and RNA processing. Melanoma inhibitory activity (MIA) has been shown to play an essential role in the progression of malignant melanoma and to influence melanoma-associated molecules and pathways in the early tumor formation steps. Interestingly, recent studies suggest that MIA is a regulator of p54(nrb). Here, we show that p54(nrb) is strongly expressed and localized in the nucleus of both melanoma cell lines and melanoma tissue samples compared with normal human melanocytes or normal skin, respectively. Furthermore, all tested melanoma cell lines revealed strong p54(nrb) promoter activity. Treatment with MIA-specific small interfering RNAs showed an influence of MIA on p54(nrb) expression on both messenger RNA (mRNA) and protein level. Knockdown of p54(nrb) protein in melanoma cell lines led to reduced proliferation rates and to a strong decrease in their migratory potential. In addition, attachment to laminin and poly-l-lysine was significantly increased. We could identify Connexin-43 (Cx-43) as a downstream target molecule of p54(nrb) as knockdown of p54(nrb) resulted in enhanced Cx-43 mRNA and protein levels. As a confirmation of these findings, melanoma cell lines showed very low Cx-43 expression levels compared with melanocytes. Our results demonstrate that p54(nrb) is highly expressed in malignant melanoma and, as a MIA target molecule, it seems to be involved in the development and progression of malignant melanoma.

  20. Melanoma: Genetic Abnormalities, Tumor Progression, Clonal Evolution and Tumor Initiating Cells.

    PubMed

    Testa, Ugo; Castelli, Germana; Pelosi, Elvira

    2017-11-20

    Melanoma is an aggressive neoplasia issued from the malignant transformation of melanocytes, the pigment-generating cells of the skin. It is responsible for about 75% of deaths due to skin cancers. Melanoma is a phenotypically and molecularly heterogeneous disease: cutaneous, uveal, acral, and mucosal melanomas have different clinical courses, are associated with different mutational profiles, and possess distinct risk factors. The discovery of the molecular abnormalities underlying melanomas has led to the promising improvement of therapy, and further progress is expected in the near future. The study of melanoma precursor lesions has led to the suggestion that the pathway of tumor evolution implies the progression from benign naevi, to dysplastic naevi, to melanoma in situ and then to invasive and metastatic melanoma. The gene alterations characterizing melanomas tend to accumulate in these precursor lesions in a sequential order. Studies carried out in recent years have, in part, elucidated the great tumorigenic potential of melanoma tumor cells. These findings have led to speculation that the cancer stem cell model cannot be applied to melanoma because, in this malignancy, tumor cells possess an intrinsic plasticity, conferring the capacity to initiate and maintain the neoplastic process to phenotypically different tumor cells.

  1. p53 prevents progression of nevi to melanoma predominantly through cell cycle regulation

    PubMed Central

    Terzian, Tamara; Torchia, Enrique C.; Dai, Daisy; Robinson, Steven E.; Murao, Kazutoshi; Stiegmann, Regan A.; Gonzalez, Victoria; Boyle, Glen M.; Powell, Marianne B.; Pollock, Pamela M.; Lozano, Guillermina; Robinson, William A.; Roop, Dennis R.; Box, Neil F.

    2011-01-01

    p53 is the central member of a critical tumor suppressor pathway in virtually all tumor types, where it is silenced mainly by missense mutations. In melanoma, p53 predominantly remains wild type, thus its role has been neglected. To study the effect of p53 on melanocyte function and melanomagenesis, we crossed the ‘high-p53’ Mdm4+/− mouse to the well-established TP-ras0/+ murine melanoma progression model. After treatment with the carcinogen dimethylbenzanthracene (DMBA), TP-ras0/+ mice on the Mdm4+/− background developed fewer tumors with a delay in the age of onset of melanomas compared to TP-ras0/+ mice. Furthermore, we observed a dramatic decrease in tumor growth, lack of metastasis with increased survival of TP-ras0/+: Mdm4+/− mice. Thus, p53 effectively prevented the conversion of small benign tumors to malignant and metastatic melanoma. p53 activation in cultured primary melanocyte and melanoma cell lines using Nutlin-3, a specific Mdm2 antagonist, supported these findings. Moreover, global gene expression and network analysis of Nutlin-3-treated primary human melanocytes indicated that cell cycle regulation through the p21WAF1/CIP1 signaling network may be the key anti-melanomagenic activity of p53. PMID:20849464

  2. miR-137 inhibits the invasion of melanoma cells through downregulation of multiple oncogenic target genes.

    PubMed

    Luo, Chonglin; Tetteh, Paul W; Merz, Patrick R; Dickes, Elke; Abukiwan, Alia; Hotz-Wagenblatt, Agnes; Holland-Cunz, Stefan; Sinnberg, Tobias; Schittek, Birgit; Schadendorf, Dirk; Diederichs, Sven; Eichmüller, Stefan B

    2013-03-01

    MicroRNAs are small noncoding RNAs that regulate gene expression and have important roles in various types of cancer. Previously, miR-137 was reported to act as a tumor suppressor in different cancers, including malignant melanoma. In this study, we show that low miR-137 expression is correlated with poor survival in stage IV melanoma patients. We identified and validated two genes (c-Met and YB1) as direct targets of miR-137 and confirmed two previously known targets, namely enhancer of zeste homolog 2 (EZH2) and microphthalmia-associated transcription factor (MITF). Functional studies showed that miR-137 suppressed melanoma cell invasion through the downregulation of multiple target genes. The decreased invasion caused by miR-137 overexpression could be phenocopied by small interfering RNA knockdown of EZH2, c-Met, or Y box-binding protein 1 (YB1). Furthermore, miR-137 inhibited melanoma cell migration and proliferation. Finally, miR-137 induced apoptosis in melanoma cell lines and decreased BCL2 levels. In summary, our study confirms that miR-137 acts as a tumor suppressor in malignant melanoma and reveals that miR-137 regulates multiple targets including c-Met, YB1, EZH2, and MITF.

  3. Detection of melanoma cells suspended in mononuclear cells and blood plasma using photoacoustic generation

    NASA Astrophysics Data System (ADS)

    Spradling, Emily M.; Viator, John A.

    2009-02-01

    Melanoma is the deadliest form of skin cancer. Although the initial malignant cells are removed, it is impossible to determine whether or not the cancer has metastasized until a secondary tumor forms that is large enough to detect with conventional imaging. Photoacoustic detection of circulating melanoma cells in the bloodstream has shown promise for early detection of metastasis that may aid in treatment of this aggressive cancer. When blood is irradiated with energy from an Nd:YAG laser at 532 nm, photoacoustic signals are created and melanoma cells can be differentiated from the surrounding cells based on waveforms produced by an oscilloscope. Before this can be used as a diagnostic technique, however, we needed to investigate several parameters. Specifically, the current technique involves the in vitro separation of blood through centrifugation to isolate and test only the white blood cell layer. Using this method, we have detected a single cultured melanoma cell among a suspension of white blood cells. However, the process could be made simpler if the plasma layer were used for detection instead of the white blood cell layer. This layer is easier to obtain after blood separation, the optical difference between plasma and melanoma cells is more pronounced in this layer than in the white blood cell layer, and the possibility that any stray red blood cells could distort the results is eliminated. Using the photoacoustic apparatus, we detected no melanoma cells within the plasma of whole blood samples spiked with cultured melanoma cells.

  4. Radiation induces an antitumour immune response to mouse melanoma.

    PubMed

    Perez, Carmen A; Fu, Allie; Onishko, Halina; Hallahan, Dennis E; Geng, Ling

    2009-12-01

    Irradiation of cancer cells can cause immunogenic death. We used mouse models to determine whether irradiation of melanoma can enhance the host antitumour immune response and function as an effective vaccination strategy, and investigated the molecular mechanisms involved in this radiation-induced response. For in vivo studies, C57BL6/J mice and the B16F0 melanoma cell line were used in a lung metastasis model, intratumoural host immune activation assays, and tumour growth delay studies. In vitro studies included a dendritic cell (DC) phagocytosis assay, detection of cell surface exposure of the protein calreticulin (CRT), and small interfering RNA (siRNA)-mediated depletion of CRT cellular levels. Irradiation of cutaneous melanomas prior to their resection resulted in more than 20-fold reduction in lung metastases after systemic challenge with untreated melanoma cells. A syngeneic vaccine derived from irradiated melanoma cells also induced adaptive immune response markers in irradiated melanoma implants. Our data indicate a trend for radiation-induced increase in melanoma cell surface exposure of CRT, which is involved in the enhanced phagocytic activity of DC against irradiated melanoma cells (VIACUC). The present study suggests that neoadjuvant irradiation of cutaneous melanoma tumours prior to surgical resection can stimulate an endogenous anti-melanoma host immune response.

  5. SWI/SNF chromatin remodeling complex is critical for the expression of microphthalmia-associated transcription factor in melanoma cells

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

    Vachtenheim, Jiri, E-mail: jivach@upn.anet.cz; Ondrusova, Lubica; Borovansky, Jan

    2010-02-12

    The microphthalmia-associated transcription factor (MITF) is required for melanocyte development, maintenance of the melanocyte-specific transcription, and survival of melanoma cells. MITF positively regulates expression of more than 25 genes in pigment cells. Recently, it has been demonstrated that expression of several MITF downstream targets requires the SWI/SNF chromatin remodeling complex, which contains one of the two catalytic subunits, Brm or Brg1. Here we show that the expression of MITF itself critically requires active SWI/SNF. In several Brm/Brg1-expressing melanoma cell lines, knockdown of Brg1 severely compromised MITF expression with a concomitant dowregulation of MITF targets and decreased cell proliferation. Although Brmmore » was able to substitute for Brg1 in maintaining MITF expression and melanoma cell proliferation, sequential knockdown of both Brm and Brg1 in 501mel cells abolished proliferation. In Brg1-null SK-MEL-5 melanoma cells, depletion of Brm alone was sufficient to abrogate MITF expression and cell proliferation. Chromatin immunoprecipitation confirmed the binding of Brg1 or Brm to the promoter of MITF. Together these results demonstrate the essential role of SWI/SNF for expression of MITF and suggest that SWI/SNF may be a promissing target in melanoma therapy.« less

  6. Identification of cells initiating human melanomas.

    PubMed

    Schatton, Tobias; Murphy, George F; Frank, Natasha Y; Yamaura, Kazuhiro; Waaga-Gasser, Ana Maria; Gasser, Martin; Zhan, Qian; Jordan, Stefan; Duncan, Lyn M; Weishaupt, Carsten; Fuhlbrigge, Robert C; Kupper, Thomas S; Sayegh, Mohamed H; Frank, Markus H

    2008-01-17

    Tumour-initiating cells capable of self-renewal and differentiation, which are responsible for tumour growth, have been identified in human haematological malignancies and solid cancers. If such minority populations are associated with tumour progression in human patients, specific targeting of tumour-initiating cells could be a strategy to eradicate cancers currently resistant to systemic therapy. Here we identify a subpopulation enriched for human malignant-melanoma-initiating cells (MMIC) defined by expression of the chemoresistance mediator ABCB5 (refs 7, 8) and show that specific targeting of this tumorigenic minority population inhibits tumour growth. ABCB5+ tumour cells detected in human melanoma patients show a primitive molecular phenotype and correlate with clinical melanoma progression. In serial human-to-mouse xenotransplantation experiments, ABCB5+ melanoma cells possess greater tumorigenic capacity than ABCB5- bulk populations and re-establish clinical tumour heterogeneity. In vivo genetic lineage tracking demonstrates a specific capacity of ABCB5+ subpopulations for self-renewal and differentiation, because ABCB5+ cancer cells generate both ABCB5+ and ABCB5- progeny, whereas ABCB5- tumour populations give rise, at lower rates, exclusively to ABCB5- cells. In an initial proof-of-principle analysis, designed to test the hypothesis that MMIC are also required for growth of established tumours, systemic administration of a monoclonal antibody directed at ABCB5, shown to be capable of inducing antibody-dependent cell-mediated cytotoxicity in ABCB5+ MMIC, exerted tumour-inhibitory effects. Identification of tumour-initiating cells with enhanced abundance in more advanced disease but susceptibility to specific targeting through a defining chemoresistance determinant has important implications for cancer therapy.

  7. Identification of cells initiating human melanomas

    PubMed Central

    Schatton, Tobias; Murphy, George F.; Frank, Natasha Y.; Yamaura, Kazuhiro; Waaga-Gasser, Ana Maria; Gasser, Martin; Zhan, Qian; Jordan, Stefan; Duncan, Lyn M.; Weishaupt, Carsten; Fuhlbrigge, Robert C.; Kupper, Thomas S.; Sayegh, Mohamed H.; Frank, Markus H.

    2012-01-01

    Tumour-initiating cells capable of self-renewal and differentiation, which are responsible for tumour growth, have been identified in human haematological malignancies1,2 and solid cancers3–6. If such minority populations are associated with tumour progression in human patients, specific targeting of tumour-initiating cells could be a strategy to eradicate cancers currently resistant to systemic therapy. Here we identify a subpopulation enriched for human malignant-melanoma-initiating cells (MMIC) defined by expression of the chemoresistance mediator ABCB5 (refs 7, 8) and show that specific targeting of this tumorigenic minority population inhibits tumour growth. ABCB5+ tumour cells detected in human melanoma patients show a primitive molecular phenotype and correlate with clinical melanoma progression. In serial human-to-mouse xenotransplantation experiments, ABCB5+ melanoma cells possess greater tumorigenic capacity than ABCB5− bulk populations and re-establish clinical tumour heterogeneity. In vivo genetic lineage tracking demonstrates a specific capacity of ABCB5+ sub-populations for self-renewal and differentiation, because ABCB5+ cancer cells generate both ABCB5+ and ABCB5− progeny, whereas ABCB5− tumour populations give rise, at lower rates, exclusively to ABCB5− cells. In an initial proof-of-principle analysis, designed to test the hypothesis that MMIC are also required for growth of established tumours, systemic administration of a monoclonal antibody directed at ABCB5, shown to be capable of inducing antibody-dependent cell-mediated cytotoxicity in ABCB5+ MMIC, exerted tumour-inhibitory effects. Identification of tumour-initiating cells with enhanced abundance in more advanced disease but susceptibility to specific targeting through a defining chemoresistance determinant has important implications for cancer therapy. PMID:18202660

  8. Detection and capture of single circulating melanoma cells using photoacoustic flowmetry

    NASA Astrophysics Data System (ADS)

    O'Brien, Christine; Mosley, Jeffrey; Goldschmidt, Benjamin S.; Viator, John A.

    2010-02-01

    Photoacoustic flowmetry has been used to detect single circulating melanoma cells in vitro. Circulating melanoma cells are those cells that travel in the blood and lymph systems to create secondary tumors and are the hallmark of metastasis. This technique involves taking blood samples from patients, separating the white blood and melanoma cells from whole blood and irradiating them with a pulsed laser in a flowmetry set up. Rapid, visible wavelength laser pulses on the order of 5 ns can induce photoacoustic waves in melanoma cells due to their melanin content, while surrounding white blood cells remain acoustically passive. We have developed a system that identifies rare melanoma cells and captures them in 50 microliter volumes using suction applied near the photoacoustic detection chamber. The 50 microliter sample is then diluted and the experiment is repeated using the new sample until only a melanoma cell remains. We have tested this system on dyed microspheres ranging in size from 300 to 500 microns. Capture of circulating melanoma cells may provide the opportunity to study metastatic cells for basic understanding of the spread of cancer and to optimize patient specific therapies.

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

    PubMed

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

    2017-06-15

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

  10. Modeling tandem AAG8-MEK inhibition in melanoma cells

    PubMed Central

    Sun, Bing; Kawahara, Masahiro; Nagamune, Teruyuki

    2014-01-01

    Drug resistance presents a challenge to the treatment of cancer patients, especially for melanomas, most of which are caused by the hyperactivation of MAPK signaling pathway. Innate or acquired drug-resistant relapse calls for the investigation of the resistant mechanisms and new anti-cancer drugs to provide implications for the ultimate goal of curative therapy. Aging-associated gene 8 (AAG8, encoded by the SIGMAR1 gene) is a chaperone protein profoundly elaborated in neurology. However, roles of AAG8 in carcinogenesis remain unclear. Herein, we discover AAG8 antagonists as new MEK inhibitors in melanoma cells and propose a novel drug combination strategy for melanoma therapy by presenting the experimental evidences. We report that specific antagonism of AAG8, efficiently suppresses melanoma cell growth and migration through, at least in part, the inactivation of the RAS-CRAF-MEK signaling pathway. We further demonstrate that melanoma cells that are resistant to AAG8 antagonist harbor refractory CRAF-MEK activity. MEK acts as a central mediator for anti-cancer effects and also for the resistance mechanism, leading to our proposal of tandem AAG8-MEK inhibition in melanoma cells. Combination of AAG8 antagonist and very low concentration of a MEK inhibitor synergistically restricts the growth of drug-resistant cells. These data collectively pinpoint AAG8 as a potential target and delineate a promising drug combination strategy for melanoma therapy. PMID:24634165

  11. Ginseng-berry-mediated gold and silver nanoparticle synthesis and evaluation of their in vitro antioxidant, antimicrobial, and cytotoxicity effects on human dermal fibroblast and murine melanoma skin cell lines

    PubMed Central

    Jiménez Pérez, Zuly Elizabeth; Mathiyalagan, Ramya; Markus, Josua; Kim, Yeon-Ju; Kang, Hyun Mi; Abbai, Ragavendran; Seo, Kwang Hoon; Wang, Dandan; Soshnikova, Veronika; Yang, Deok Chun

    2017-01-01

    There has been a growing interest in the design of environmentally affable and biocompatible nanoparticles among scientists to find novel and safe biomaterials. Panax ginseng Meyer berries have unique phytochemical profile and exhibit beneficial pharmacological activities such as antihyperglycemic, antiobesity, antiaging, and antioxidant properties. A comprehensive study of the biologically active compounds in ginseng berry extract (GBE) and the ability of ginseng berry (GB) as novel material for the biosynthesis of gold nanoparticles (GBAuNPs) and silver nanoparticles (GBAgNPs) was conducted. In addition, the effects of GBAuNPs and GBAgNPs on skin cell lines for further potential biological applications are highlighted. GBAuNPs and GBAgNPs were synthesized using aqueous GBE as a reducing and capping agent. The synthesized nanoparticles were characterized for their size, morphology, and crystallinity. The nanoparticles were evaluated for antioxidant, anti-tyrosinase, antibacterial, and cytotoxicity activities and for morphological changes in human dermal fibroblast and murine melanoma skin cell lines. The phytochemicals contained in GBE effectively reduced and capped gold and silver ions to form GBAuNPs and GBAgNPs. The optimal synthesis conditions (ie, temperature and v/v % of GBE) and kinetics were investigated. Polysaccharides and phenolic compounds present in GBE were suggested to be responsible for stabilization and functionalization of nanoparticles. GBAuNPs and GBAgNPs showed increased scavenging activity against 2,2-diphenyl-1-picrylhydrazyl free radicals compared to GBE. GBAuNPs and GBAgNPs effectively inhibited mushroom tyrosinase, while GBAgNPs showed antibacterial activity against Escherichia coli and Staphylococcus aureus. In addition, GBAuNPs were nontoxic to human dermal fibroblast and murine melanoma cell lines, and GBAgNPs showed cytotoxic effect on murine melanoma cell lines. The current results evidently suggest that GBAgNPs can act as potential

  12. Melanoma: Genetic Abnormalities, Tumor Progression, Clonal Evolution and Tumor Initiating Cells

    PubMed Central

    Castelli, Germana; Pelosi, Elvira

    2017-01-01

    Melanoma is an aggressive neoplasia issued from the malignant transformation of melanocytes, the pigment-generating cells of the skin. It is responsible for about 75% of deaths due to skin cancers. Melanoma is a phenotypically and molecularly heterogeneous disease: cutaneous, uveal, acral, and mucosal melanomas have different clinical courses, are associated with different mutational profiles, and possess distinct risk factors. The discovery of the molecular abnormalities underlying melanomas has led to the promising improvement of therapy, and further progress is expected in the near future. The study of melanoma precursor lesions has led to the suggestion that the pathway of tumor evolution implies the progression from benign naevi, to dysplastic naevi, to melanoma in situ and then to invasive and metastatic melanoma. The gene alterations characterizing melanomas tend to accumulate in these precursor lesions in a sequential order. Studies carried out in recent years have, in part, elucidated the great tumorigenic potential of melanoma tumor cells. These findings have led to speculation that the cancer stem cell model cannot be applied to melanoma because, in this malignancy, tumor cells possess an intrinsic plasticity, conferring the capacity to initiate and maintain the neoplastic process to phenotypically different tumor cells. PMID:29156643

  13. Combined pitavastatin and dacarbazine treatment activates apoptosis and autophagy resulting in synergistic cytotoxicity in melanoma cells.

    PubMed

    Al-Qatati, Abeer; Aliwaini, Saeb

    2017-12-01

    Melanoma is an aggressive skin cancer and its incidence is increasing faster than any other type of cancer. Whilst dacarbazine (DTIC) is the standard chemotherapy for metastatic melanoma, it has limited success. Statins, including pitavastatin, have been demonstrated to have a range of anti-cancer effects in a number of human cancer cell lines. The present study therefore explored the anti-cancer activity of combined DTIC and pitavastatin in A375 and WM115 human melanoma cells. Cell survival assays demonstrated that combined DTIC and pitavastatin treatment resulted in synergistic cell death. Cell cycle analyses further revealed that this combined treatment resulted in a G1 cell cycle arrest, as well as a sub-G1 population, indicative of apoptosis. Activation of apoptosis was confirmed by Annexin V-fluorescein isothiocyanate/propidium iodide double-staining and an increase in the levels of active caspase 3 and cleaved poly (ADP-ribose) polymerase. Furthermore, it was demonstrated that apoptosis occurs through the intrinsic pathway, evident from the release of cytochrome c. Finally, combined DTIC and pitavastatin treatment was demonstrated to also activate autophagy as part of a cell death mechanism. The present study provides novel evidence to suggest that the combined treatment of DTIC and pitavastatin may be effective in the treatment of melanoma.

  14. Combined pitavastatin and dacarbazine treatment activates apoptosis and autophagy resulting in synergistic cytotoxicity in melanoma cells

    PubMed Central

    Al-Qatati, Abeer; Aliwaini, Saeb

    2017-01-01

    Melanoma is an aggressive skin cancer and its incidence is increasing faster than any other type of cancer. Whilst dacarbazine (DTIC) is the standard chemotherapy for metastatic melanoma, it has limited success. Statins, including pitavastatin, have been demonstrated to have a range of anti-cancer effects in a number of human cancer cell lines. The present study therefore explored the anti-cancer activity of combined DTIC and pitavastatin in A375 and WM115 human melanoma cells. Cell survival assays demonstrated that combined DTIC and pitavastatin treatment resulted in synergistic cell death. Cell cycle analyses further revealed that this combined treatment resulted in a G1 cell cycle arrest, as well as a sub-G1 population, indicative of apoptosis. Activation of apoptosis was confirmed by Annexin V-fluorescein isothiocyanate/propidium iodide double-staining and an increase in the levels of active caspase 3 and cleaved poly (ADP-ribose) polymerase. Furthermore, it was demonstrated that apoptosis occurs through the intrinsic pathway, evident from the release of cytochrome c. Finally, combined DTIC and pitavastatin treatment was demonstrated to also activate autophagy as part of a cell death mechanism. The present study provides novel evidence to suggest that the combined treatment of DTIC and pitavastatin may be effective in the treatment of melanoma. PMID:29344241

  15. IgG1-iS18 impedes the adhesive and invasive potential of early and late stage malignant melanoma cells.

    PubMed

    Munien, Carmelle; Rebelo, Thalia M; Ferreira, Eloise; Weiss, Stefan F T

    2017-02-15

    The 37kDa/67kDa laminin receptor (LRP/LR) is a non-integrin laminin receptor which is overexpressed in tumorigenic cells and supports progression of cancer via promoting metastasis, angiogenesis and telomerase activity and impediment of apoptosis. The present study investigates the role of LRP/LR on the metastatic potential of early (A375) and late (A375SM) stage malignant melanoma cells. Flow cytometry revealed that both early and late stage malignant melanoma cells display high levels of LRP/LR on their cell surface. Flow cytometry and western blot analysis showed that late stage malignant melanoma cells display significantly higher total and cell surface LRP/LR levels in comparison to early stage malignant melanoma cells and the poorly invasive breast cancer (MCF-7) control cell line. Targeting LRP/LR using the LRP/LR specific antibody IgG1-iS18 resulted in a significant reduction of the adhesive potential to laminin-1 and the invasive potential through the 'ECM-simulating' Matrigel™ of both early and late stage malignant melanoma cells. Furthermore, Pearson's correlation coefficient confirmed that increased LRP levels correlate with the increased invasive and adhesive potential in early and late stage melanoma cells. Thus, blocking LRP/LR using the IgG1-iS18 antibody may therefore be a promising therapeutic strategy for early and late stage malignant melanoma treatment. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Immunoregulatory protein B7-H3 promotes growth and decreases sensitivity to therapy in metastatic melanoma cells.

    PubMed

    Flem-Karlsen, Karine; Tekle, Christina; Andersson, Yvonne; Flatmark, Kjersti; Fodstad, Øystein; Nunes-Xavier, Caroline E

    2017-09-01

    B7-H3 (CD276) belongs to the B7 family of immunoregulatory proteins and has been implicated in cancer progression and metastasis. In this study, we found that metastatic melanoma cells with knockdown expression of B7-H3 showed modest decrease in proliferation and glycolytic capacity and were more sensitive to dacarbazine (DTIC) chemotherapy and small-molecule inhibitors targeting MAP kinase (MAPK) and AKT/mTOR pathways: vemurafenib (PLX4032; BRAF inhibitor), binimetinib (MEK-162; MEK inhibitor), everolimus (RAD001; mTOR inhibitor), and triciribidine (API-2; AKT inhibitor). Similar effects were observed in melanoma cells in the presence of an inhibitory B7-H3 monoclonal antibody, while the opposite was seen in B7-H3-overexpressing cells. Further, combining B7-H3 inhibition with small-molecule inhibitors resulted in significantly increased antiproliferative effect in melanoma cells, as well as in BRAF V 600E mutated cell lines derived from patient biopsies. Our findings indicate that targeting B7-H3 may be a novel alternative to improve current therapy of metastatic melanoma. © 2017 The Authors Pigment Cell & Melonoma Research Published by John Wiley & Sons Ltd.

  17. Heat Shock Protein-90 Inhibitors Enhance Antigen Expression on Melanomas and Increase T Cell Recognition of Tumor Cells

    PubMed Central

    Haggerty, Timothy J.; Dunn, Ian S.; Rose, Lenora B.; Newton, Estelle E.; Pandolfi, Franco; Kurnick, James T.

    2014-01-01

    In an effort to enhance antigen-specific T cell recognition of cancer cells, we have examined numerous modulators of antigen-expression. In this report we demonstrate that twelve different Hsp90 inhibitors (iHsp90) share the ability to increase the expression of differentiation antigens and MHC Class I antigens. These iHsp90 are active in several molecular and cellular assays on a series of tumor cell lines, including eleven human melanomas, a murine B16 melanoma, and two human glioma-derived cell lines. Intra-cytoplasmic antibody staining showed that all of the tested iHsp90 increased expression of the melanocyte differentiation antigens Melan-A/MART-1, gp100, and TRP-2, as well as MHC Class I. The gliomas showed enhanced gp100 and MHC staining. Quantitative analysis of mRNA levels showed a parallel increase in message transcription, and a reporter assay shows induction of promoter activity for Melan-A/MART-1 gene. In addition, iHsp90 increased recognition of tumor cells by T cells specific for Melan-A/MART-1. In contrast to direct Hsp90 client proteins, the increased levels of full-length differentiation antigens that result from iHsp90 treatment are most likely the result of transcriptional activation of their encoding genes. In combination, these results suggest that iHsp90 improve recognition of tumor cells by T cells specific for a melanoma-associated antigen as a result of increasing the expressed intracellular antigen pool available for processing and presentation by MHC Class I, along with increased levels of MHC Class I itself. As these Hsp90 inhibitors do not interfere with T cell function, they could have potential for use in immunotherapy of cancer. PMID:25503774

  18. An autophagy-driven pathway of ATP secretion supports the aggressive phenotype of BRAFV600E inhibitor-resistant metastatic melanoma cells.

    PubMed

    Martin, Shaun; Dudek-Peric, Aleksandra M; Garg, Abhishek D; Roose, Heleen; Demirsoy, Seyma; Van Eygen, Sofie; Mertens, Freya; Vangheluwe, Peter; Vankelecom, Hugo; Agostinis, Patrizia

    2017-09-02

    The ingrained capacity of melanoma cells to rapidly evolve toward an aggressive phenotype is manifested by their increased ability to develop drug-resistance, evident in the case of vemurafenib, a therapeutic-agent targeting BRAF V600E . Previous studies indicated a tight correlation between heightened melanoma-associated macroautophagy/autophagy and acquired Vemurafenib resistance. However, how this vesicular trafficking pathway supports Vemurafenib resistance remains unclear. Here, using isogenic human and murine melanoma cell lines of Vemurafenib-resistant and patient-derived melanoma cells with primary resistance to the BRAF V600E inhibitor, we found that the enhanced migration and invasion of the resistant melanoma cells correlated with an enhanced autophagic capacity and autophagosome-mediated secretion of ATP. Extracellular ATP (eATP) was instrumental for the invasive phenotype and the expansion of a subset of Vemurafenib-resistant melanoma cells. Compromising the heightened autophagy in these BRAF V600E inhibitor-resistant melanoma cells through the knockdown of different autophagy genes (ATG5, ATG7, ULK1), reduced their invasive and eATP-secreting capacity. Furthermore, eATP promoted the aggressive nature of the BRAF V600E inhibitor-resistant melanoma cells by signaling through the purinergic receptor P2RX7. This autophagy-propelled eATP-dependent autocrine-paracrine pathway supported the maintenance and expansion of a drug-resistant melanoma phenotype. In conclusion, we have identified an autophagy-driven response that relies on the secretion of ATP to drive P2RX7-based migration and expansion of the Vemurafenib-resistant phenotype. This emphasizes the potential of targeting autophagy in the treatment and management of metastatic melanoma.

  19. Adoptive Cell Therapy with Tumor-Infiltrating Lymphocytes in Advanced Melanoma Patients

    PubMed Central

    Saint-Jean, Mélanie; Volteau, Christelle; Quéreux, Gaëlle; Peuvrel, Lucie; Brocard, Anabelle; Saiagh, Soraya; Nguyen, Jean-Michel; Bedane, Christophe; Basset-Seguin, Nicole

    2018-01-01

    Immunotherapy for melanoma includes adoptive cell therapy with autologous tumor-infiltrating lymphocytes (TILs). This monocenter retrospective study was undertaken to evaluate the efficacy and safety of this treatment of patients with advanced melanoma. All advanced melanoma patients treated with TILs using the same TIL expansion methodology and same treatment interleukin-2 (IL-2) regimen between 2009 and 2012 were included. After sterile intralesional excision of a cutaneous or subcutaneous metastasis, TILs were produced according to a previously described method and then infused into the patient who also received a complementary subcutaneous IL-2 regimen. Nine women and 1 man were treated for unresectable stage IIIC (n = 4) or IV (n = 6) melanoma. All but 1 patient with unresectable stage III melanoma (1st line) had received at least 2 previous treatments, including anti-CTLA-4 antibody for 4. The number of TILs infused ranged from 0.23 × 109 to 22.9 × 109. Regarding safety, no serious adverse effect was reported. Therapeutic responses included a complete remission, a partial remission, 2 stabilizations, and 6 progressions. Among these 4 patients with clinical benefit, 1 is still alive with 9 years of follow-up and 1 died from another cause after 8 years of follow-up. Notably, patients treated with high percentages of CD4 + CD25 + CD127lowFoxp3+ T cells among their TILs had significantly shorter OS. The therapeutic effect of combining TILs with new immunotherapies needs further investigation. PMID:29750176

  20. Modeling tandem AAG8-MEK inhibition in melanoma cells.

    PubMed

    Sun, Bing; Kawahara, Masahiro; Nagamune, Teruyuki

    2014-06-01

    Drug resistance presents a challenge to the treatment of cancer patients, especially for melanomas, most of which are caused by the hyperactivation of MAPK signaling pathway. Innate or acquired drug-resistant relapse calls for the investigation of the resistant mechanisms and new anti-cancer drugs to provide implications for the ultimate goal of curative therapy. Aging-associated gene 8 (AAG8, encoded by the SIGMAR1 gene) is a chaperone protein profoundly elaborated in neurology. However, roles of AAG8 in carcinogenesis remain unclear. Herein, we discover AAG8 antagonists as new MEK inhibitors in melanoma cells and propose a novel drug combination strategy for melanoma therapy by presenting the experimental evidences. We report that specific antagonism of AAG8, efficiently suppresses melanoma cell growth and migration through, at least in part, the inactivation of the RAS-CRAF-MEK signaling pathway. We further demonstrate that melanoma cells that are resistant to AAG8 antagonist harbor refractory CRAF-MEK activity. MEK acts as a central mediator for anti-cancer effects and also for the resistance mechanism, leading to our proposal of tandem AAG8-MEK inhibition in melanoma cells. Combination of AAG8 antagonist and very low concentration of a MEK inhibitor synergistically restricts the growth of drug-resistant cells. These data collectively pinpoint AAG8 as a potential target and delineate a promising drug combination strategy for melanoma therapy. © 2014 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  1. The Role of Adenosine A2BR in Metastatic Melanoma

    DTIC Science & Technology

    2017-07-01

    100% complete. ACURO approval to perform animal studies was obtained July 2016. Specific Aim 1, Subtask 2: 100% complete. Use CRISPR /Cas9 technology...immune cell interactions, the first objective was to use the CRISPR Cas9 system to knock out A2BR expression in melanoma cell lines. Melanoma cell lines...and sgRNA3 to work but sgRNA2 would not be as efficient. We considered commercially available constructs to potentially improve the CRISPR knock

  2. Comparing the efficacy of photodynamic and sonodynamic therapy in non-melanoma and melanoma skin cancer.

    PubMed

    McEwan, Conor; Nesbitt, Heather; Nicholas, Dean; Kavanagh, Oisin N; McKenna, Kevin; Loan, Philip; Jack, Iain G; McHale, Anthony P; Callan, John F

    2016-07-01

    Sonodynamic therapy (SDT) involves the activation of a non-toxic sensitiser drug using low-intensity ultrasound to produce cytotoxic reactive oxygen species (ROS). Given the low tissue attenuation of ultrasound, SDT provides a significant benefit over the more established photodynamic therapy (PDT) as it enables activation of sensitisers at a greater depth within human tissue. In this manuscript, we compare the efficacy of aminolevulinic acid (ALA) mediated PDT and SDT in a squamous cell carcinoma (A431) cell line as well as the ability of these treatments to reduce the size of A431 ectopic tumours in mice. Similarly, the relative cytotoxic ability of Rose Bengal mediated PDT and SDT was investigated in a B16-melanoma cell line and also in a B16 ectopic tumour model. The results reveal no statistically significant difference in efficacy between ALA mediated PDT or SDT in the non-melanoma model while Rose Bengal mediated SDT was significantly more efficacious than PDT in the melanoma model. This difference in efficacy was, at least in part, attributed to the dark pigmentation of the melanoma cells that effectively filtered the excitation light preventing it from activating the sensitiser while the use of ultrasound circumvented this problem. These results suggest SDT may provide a better outcome than PDT when treating highly pigmented cancerous skin lesions. Copyright © 2016. Published by Elsevier Ltd.

  3. [Endocrine factors influencing melanoma progression].

    PubMed

    Dobos, Judit

    2009-03-01

    According to recent findings that beside cancers traditionally considered as hormone-dependent, several other tumor types show different behavior in the two sexes, indicating the possible role of endocrine factors in the course of these diseases. The possibility that endocrine factors may influence the clinical course of human malignant melanoma is suggested by the higher survival rate in premenopausal vs. postmenopausal women or men of any ages. However, investigations on the sex hormone receptor status of human cutaneous melanomas and experiments attempting to support the epidemiological results yielded conflicting results. In our human melanoma cell lines we failed to detect steroid receptors at protein level, while quantitative PCR demonstrated that their mRNA expression level was orders of magnitude lower compared to the positive control cell lines. Sex hormones did not influence the in vitro features of the human melanoma cells considerably. On the other hand, glucocorticoid receptor was present both at mRNA and protein level, although dexamethasone was effective in vitro only at high doses. Our previous experiments showed that intrasplenic injection of human melanoma cells resulted in a significantly higher number of liver colonies in male than in female SCID mice. We now show that this difference evolves during the first day. After injection into the tail vein we did not observe gender-dependent difference in the efficiency of pulmonary colonization. Examining the pattern of metastasis formation after intracardiac injection, we have found differences between the two sexes in the incidence or number of colonies only in the case of the liver but not in other organs. We concluded that the observed phenomenon is specific to the liver; therefore we investigated the effects of 2-methoxyestradiol, an endogenous metabolite of estradiol produced mainly in the liver, with an estrogen receptor-independent antitumor activity. 2ME2 effectively inhibited melanoma cell

  4. Tumor cell cholesterol depletion and V-ATPase inhibition as an inhibitory mechanism to prevent cell migration and invasiveness in melanoma.

    PubMed

    Costa, Gildeíde Aparecida; de Souza, Sávio Bastos; da Silva Teixeira, Layz Ribeiro; Okorokov, Lev A; Arnholdt, Andrea Cristina Vetö; Okorokova-Façanha, Anna L; Façanha, Arnoldo Rocha

    2018-03-01

    V-ATPase interactions with cholesterol enriched membrane microdomains have been related to metastasis in a variety of cancers, but the underlying mechanism remains at its beginnings. It has recently been reported that the inhibition of this H + pump affects cholesterol mobilization to the plasma membrane. Inhibition of melanoma cell migration and invasiveness was assessed by wound healing and Transwell assays in murine cell lines (B16F10 and Melan-A). V-ATPase activity was measured in vitro by ATP hydrolysis and H + transport in membrane vesicles, and intact cell H + fluxes were measured by using a non-invasive Scanning Ion-selective Electrode Technique (SIET). Cholesterol depletion by 5mM MβCD was found to be inhibitory to the hydrolytic and H + pumping activities of the V-ATPase of melanoma cell lines, as well as to the migration and invasiveness capacities of these cells. Nearly the same effects were obtained using concanamycin A, a specific inhibitor of V-ATPase, which also promoted a decrease of the H + efflux in live cells at the same extent of MβCD. We found that cholesterol depletion significantly affects the V-ATPase activity and the initial metastatic processes following a profile similar to those observed in the presence of the V-ATPase specific inhibitor, concanamycin. The results shed new light on the functional role of the interactions between V-ATPases and cholesterol-enriched microdomains of cell membranes that contribute with malignant phenotypes in melanoma. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Mast cells promote melanoma colonization of lungs.

    PubMed

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

    2016-10-18

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

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

    NASA Astrophysics Data System (ADS)

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

    1999-02-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  8. HTB140 melanoma cells under proton irradiation and/or alkylating agents

    NASA Astrophysics Data System (ADS)

    Korićanac, L.; Petrović, I.; Privitera, G.; Cuttone, G.; Ristić-Fira, A.

    2007-09-01

    Chemoresistance is a major problem in the treatment of malignant melanoma. The mainstay of treatment for melanoma is the DNA-alkylating agent dacarbazine (DTIC). Fotemustine (FM), a member of the chloroethylnitrosourea group of alkylating agents, has also demonstrated significant antitumor effects in malignant melanoma. However, the intrinsic and acquired resistance of melanoma limits the clinical application of these drugs. Melanomas are also extremely radioresistant. With the objective of enhancing growth inhibition of melanoma cells, combined treatments of FM or DTIC with proton irradiation have been investigated. These effects were studied on HTB140 melanoma cell viability and proliferation. Cells exposed to treatment with FM and protons have shown inhibition of cell growth and significant reduction of proliferation capacity compared to single irradiation or drug treatment. Treatment with DTIC and protons has shown improved growth inhibition compared to appropriate single drug treatment, while the effects of single proton irradiation have been the most pronounced.

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

    PubMed

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

    2003-04-01

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

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

    PubMed

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

    2007-12-15

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

  11. Assaying Wnt5A-mediated Invasion in Melanoma Cells

    PubMed Central

    O'Connell, Michael P.; French, Amanda D.; Leotlela, Poloko D.; Weeraratna, Ashani T.

    2009-01-01

    Wnt5A has been implicated in melanoma metastasis, and the progression of other cancers including pancreatic, gastric, prostate and lung cancers. Assays to test motility and invasion include both in vivo assays, and in vitro assays. The former assays include the use of tail vein or footpad injections of metastatic cells, and are often laborious and expensive. In vitro invasion assays provide quick readouts that can help to establish conditions that either activate or inhibit melanoma cell motility, and to assess whether the conditions in question are worth translating into an in vivo model. Here we describe two standard methods for assaying motility and invasion in vitro including wound healing assays and Matrigel invasion assays (Boyden chamber assays). In addition, we and several other laboratories have previously shown that melanoma cells require MMP-2 for their invasion, and have recently shown that Wnt5A treatment can increase the levels of this enzyme in melanoma cells, as demonstrated by gelatin zymography. The use of these techniques can help to assess the migratory capacity of melanoma cells in response to Wnt treatment. PMID:19099260

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

    PubMed

    Zdybel, Magdalena; Chodurek, Ewa; Pilawa, Barbara

    2014-01-01

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

  13. BRAF and MEK inhibitor therapy eliminates nestin expressing melanoma cells in human tumors.

    PubMed

    Doxie, Deon B; Greenplate, Allison R; Gandelman, Jocelyn S; Diggins, Kirsten E; Roe, Caroline E; Dahlman, Kimberly B; Sosman, Jeffrey A; Kelley, Mark C; Irish, Jonathan M

    2018-05-19

    Little is known about the in vivo impacts of targeted therapy on melanoma cell abundance and protein expression. Here, 21 antibodies were added to an established melanoma mass cytometry panel to measure 32 cellular features, distinguish malignant cells, and characterize dabrafenib and trametinib responses in BRAF V 600mut melanoma. Tumor cells were biopsied before neoadjuvant therapy and compared to cells surgically resected from the same site after 4 weeks of therapy. Approximately 50,000 cells per tumor were characterized by mass cytometry and computational tools t-SNE/viSNE, FlowSOM, and MEM. The resulting single cell view of melanoma treatment response revealed initially heterogeneous melanoma tumors were consistently cleared of Nestin expressing melanoma cells. Melanoma cells subsets that persisted to week 4 were heterogeneous but expressed SOX2 or SOX10 proteins and specifically lacked surface expression of MHC I proteins by MEM analysis. Traditional histology imaging of tissue microarrays from the same tumors confirmed mass cytometry results, including persistence of NES- SOX10+ S100β+ melanoma cells. This quantitative single cell view of melanoma treatment response revealed protein features of malignant cells that are not eliminated by targeted therapy. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  14. SOX2 regulates self-renewal and tumorigenicity of human melanoma-initiating cells.

    PubMed

    Santini, R; Pietrobono, S; Pandolfi, S; Montagnani, V; D'Amico, M; Penachioni, J Y; Vinci, M C; Borgognoni, L; Stecca, B

    2014-09-18

    Melanoma is one of the most aggressive types of human cancer, characterized by enhanced heterogeneity and resistance to conventional therapy at advanced stages. We and others have previously shown that HEDGEHOG-GLI (HH-GLI) signaling is required for melanoma growth and for survival and expansion of melanoma-initiating cells (MICs). Recent reports indicate that HH-GLI signaling regulates a set of genes typically expressed in embryonic stem cells, including SOX2 (sex-determining region Y (SRY)-Box2). Here we address the function of SOX2 in human melanomas and MICs and its interaction with HH-GLI signaling. We find that SOX2 is highly expressed in melanoma stem cells. Knockdown of SOX2 sharply decreases self-renewal in melanoma spheres and in putative melanoma stem cells with high aldehyde dehydrogenase activity (ALDH(high)). Conversely, ectopic expression of SOX2 in melanoma cells enhances their self-renewal in vitro. SOX2 silencing also inhibits cell growth and induces apoptosis in melanoma cells. In addition, depletion of SOX2 progressively abrogates tumor growth and leads to a significant decrease in tumor-initiating capability of ALDH(high) MICs upon xenotransplantation, suggesting that SOX2 is required for tumor initiation and for continuous tumor growth. We show that SOX2 is regulated by HH signaling and that the transcription factors GLI1 and GLI2, the downstream effectors of HH-GLI signaling, bind to the proximal promoter region of SOX2 in primary melanoma cells. In functional studies, we find that SOX2 function is required for HH-induced melanoma cell growth and MIC self-renewal in vitro. Thus SOX2 is a critical factor for self-renewal and tumorigenicity of MICs and an important mediator of HH-GLI signaling in melanoma. These findings could provide the basis for novel therapeutic strategies based on the inhibition of SOX2 for the treatment of a subset of human melanomas.

  15. Targeted Silencing of MART-1 Gene Expression by RNA Interference Enhances the Migration Ability of Uveal Melanoma Cells

    PubMed Central

    Zhang, Yidan; Jia, Renbing; Wang, Jing; Xu, Xiaofang; Yao, Yuting; Ge, Shengfan; Fan, Xianqun

    2013-01-01

    Uveal melanoma (UM) is the most common primary intraocular malignancy and the leading potentially fatal primary intraocular disease in adults. Melanoma antigen recognized by T-cells (MART-1) has been studied extensively as a clinically important diagnostic marker for melanoma, however, its biological function remains unclear. In the present study, the UM cell line SP6.5, which showed a high level of MART-1 expression, was subjected to small interfering RNA-mediated silencing of MART-1. Silencing of MART-1 expression increased the migration ability of SP6.5 cells and down-regulated the expression of the metastasis suppressor NM23. Our results suggest that MART-1 is a candidate target for the development of therapeutic strategies for UM and in particular for the suppression of metastasis associated with this malignancy. PMID:23877836

  16. A Novel Therapy for Melanoma Developed in Mice: Transformation of Melanoma into Dendritic Cells with Listeria monocytogenes

    PubMed Central

    Bronchalo-Vicente, Lucia; Rodriguez-Del Rio, Estela; Freire, Javier; Calderon-Gonzalez, Ricardo; Frande-Cabanes, Elisabet; Gomez-Roman, Jose Javier; Fernández-Llaca, Hector; Yañez-Diaz, Sonsoles; Alvarez-Dominguez, Carmen

    2015-01-01

    Listeria monocytogenes is a gram-positive bacteria and human pathogen widely used in cancer immunotherapy because of its capacity to induce a specific cytotoxic T cell response in tumours. This bacterial pathogen strongly induces innate and specific immunity with the potential to overcome tumour induced tolerance and weak immunogenicity. Here, we propose a Listeria based vaccination for melanoma based in its tropism for these tumour cells and its ability to transform in vitro and in vivo melanoma cells into matured and activated dendritic cells with competent microbicidal and antigen processing abilities. This Listeria based vaccination using low doses of the pathogen caused melanoma regression by apoptosis as well as bacterial clearance. Vaccination efficacy is LLO dependent and implies the reduction of LLO-specific CD4+ T cell responses, strong stimulation of innate pro-inflammatory immune cells and a prevalence of LLO-specific CD8+ T cells involved in tumour regression and Listeria elimination. These results support the use of low doses of pathogenic Listeria as safe melanoma therapeutic vaccines that do not require antibiotics for bacterial removal. PMID:25760947

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

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

  18. BNIP3 contributes to the glutamine-driven aggressive behavior of melanoma cells.

    PubMed

    Vara-Perez, Monica; Maes, Hannelore; Van Dingenen, Sarah; Agostinis, Patrizia

    2018-06-01

    Aerobic glycolysis (Warburg effect) is used by cancer cells to fuel tumor growth. Interestingly, metastatic melanoma cells rely on glutaminolysis rather than aerobic glycolysis for their bioenergetic needs through the tricarboxylic acid cycle. Here, we compared the effects of glucose or glutamine on melanoma cell proliferation, migration and oxidative phosphorylation in vitro. We found that glutamine-driven melanoma cell's aggressive traits positively correlated with increased expression of HIF1α and its pro-autophagic target BNIP3. BNIP3 silencing reduced glutamine-mediated effects on melanoma cell growth, migration and bioenergetics. Hence, BNIP3 is a vital component of the mitochondria quality control required for glutamine-driven melanoma aggressiveness.

  19. Epac1 increases migration of endothelial cells and melanoma cells via FGF2-mediated paracrine signaling

    PubMed Central

    Baljinnyam, Erdene; Umemura, Masanari; Chuang, Christine; De Lorenzo, Mariana S; Iwatsubo, Mizuka; Chen, Suzie; Goydos, James S; Ishikawa, Yoshihiro; Whitelock, John M; Iwatsubo, Kousaku

    2014-01-01

    Fibroblast growth factor (FGF2) regulates endothelial and melanoma cell migration. The binding of FGF2 to its receptor requires N-sulfated heparan sulfate (HS) glycosamine. We have previously reported that Epac1, an exchange protein activated by cAMP, increases N-sulfation of HS in melanoma. Therefore, we examined whether Epac1 regulates FGF2-mediated cell–cell communication. Conditioned medium (CM) of melanoma cells with abundant expression of Epac1 increased migration of human umbilical endothelial cells (HUVEC) and melanoma cells with poor expression of Epac1. CM-induced increase in migration was inhibited by antagonizing FGF2, by the removal of HS and by the knockdown of Epac1. In addition, knockdown of Epac1 suppressed the binding of FGF2 to FGF receptor in HUVEC, and in vivo angiogenesis in melanoma. Furthermore, knockdown of Epac1 reduced N-sulfation of HS chains attached to perlecan, a major secreted type of HS proteoglycan that mediates the binding of FGF2 to FGF receptor. These data suggested that Epac1 in melanoma cells regulates melanoma progression via the HS–FGF2-mediated cell–cell communication. PMID:24725364

  20. Frequent Nuclear/Cytoplasmic Localization of β-Catenin without Exon 3 Mutations in Malignant Melanoma

    PubMed Central

    Rimm, David L.; Caca, Karel; Hu, Gang; Harrison, Frank B.; Fearon, Eric R.

    1999-01-01

    β-Catenin has a critical role in E-cadherin-mediated cell-cell adhesion, and it also functions as a downstream signaling molecule in the wnt pathway. Mutations in the putative glycogen synthase kinase 3β phosphorylation sites near the β-catenin amino terminus have been found in some cancers and cancer cell lines. The mutations render β-catenin resistant to regulation by a complex containing the glycogen synthase kinase 3β, adenomatous polyposis coli, and axin proteins. As a result, β-catenin accumulates in the cytosol and nucleus and activates T-cell factor/lymphoid enhancing factor transcription factors. Previously, 6 of 27 melanoma cell lines were found to have β-catenin exon 3 mutations affecting the N-terminal phosphorylation sites (Rubinfeld B, Robbins P, Elgamil M, Albert I, Porfiri E, Polakis P: Stabilization of beta-catenin by genetic defects in melanoma cell lines. Science 1997, 275:1790–1792). To assess the role of β-catenin defects in primary melanomas, we undertook immunohistochemical and DNA sequencing studies in 65 melanoma specimens. Nuclear and/or cytoplasmic localization of β-catenin, a potential indicator of wnt pathway activation, was seen focally within roughly one third of the tumors, though a clonal somatic mutation in β-catenin was found in only one case (codon 45 Ser→Pro). Our findings demonstrate that β-catenin mutations are rare in primary melanoma, in contrast to the situation in melanoma cell lines. Nonetheless, activation of β-catenin, as indicated by its nuclear and/or cytoplasmic localization, appears to be frequent in melanoma, and in some cases, it may reflect focal and transient activation of the wnt pathway within the tumor. PMID:10027390

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

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

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

    2007-03-23

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

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

    PubMed Central

    Pal, Harish Chandra; Sharma, Samriti; Strickland, Leah Ray; Katiyar, Santosh K.; Ballestas, Mary E.; Athar, Mohammad; Elmets, Craig A.; Afaq, Farrukh

    2014-01-01

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

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

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

    Ungerer, Christopher; Doberstein, Kai; Buerger, Claudia

    2010-10-22

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

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

    PubMed Central

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

    2009-01-01

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

  5. Priming Endothelial Cells With a Melanoma-Derived Extracellular Matrix Triggers the Activation of αvβ3/VEGFR2 Axis.

    PubMed

    Helal-Neto, Edward; Brandão-Costa, Renata M; Saldanha-Gama, Roberta; Ribeiro-Pereira, Cristiane; Midlej, Victor; Benchimol, Marlene; Morandi, Verônica; Barja-Fidalgo, Christina

    2016-11-01

    The unique composition of tumor-produced extracellular matrix (ECM) can be a determining factor in changing the profile of endothelial cells in the tumor microenvironment. As the main receptor for ECM proteins, integrins can activate a series of signaling pathways related to cell adhesion, migration, and differentiation of endothelial cells that interact with ECM proteins. We studied the direct impact of the decellularized ECM produced by a highly metastatic human melanoma cell line (MV3) on the activation of endothelial cells and identified the intracellular signaling pathways associated with cell differentiation. Our data show that compared to the ECM derived from a human melanocyte cell line (NGM-ECM), ECM produced by a melanoma cell line (MV3-ECM) is considerably different in ultrastructural organization and composition and possesses a higher content of tenascin-C and laminin and a lower expression of fibronectin. When cultured directly on MV3-ECM, endothelial cells change morphology and show increased adhesion, migration, proliferation, and tubulogenesis. Interaction of endothelial cells with MV3-ECM induces the activation of integrin signaling, increasing FAK phosphorylation and its association with Src, which activates VEGFR2, potentiating the receptor response to VEGF. The blockage of αvβ3 integrin inhibited the FAK-Src association and VEGFR activation, thus reducing tubulogenesis. Together, our data suggest that the interaction of endothelial cells with the melanoma-ECM triggers integrin-dependent signaling, leading to Src pathway activation that may potentiate VEGFR2 activation and up-regulate angiogenesis. J. Cell. Physiol. 231: 2464-2473, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  6. Chronic mild stress facilitates melanoma tumor growth in mouse lines selected for high and low stress-induced analgesia.

    PubMed

    Ragan, Agnieszka R; Lesniak, Anna; Bochynska-Czyz, Marta; Kosson, Anna; Szymanska, Hanna; Pysniak, Kazimiera; Gajewska, Marta; Lipkowski, Andrzej W; Sacharczuk, Mariusz

    2013-09-01

    Both chronic stress conditions and hyperergic reaction to environmental stress are known to enhance cancer susceptibility. We described two mouse lines that displayed high (HA) and low (LA) swim stress-induced analgesia (SSIA) to investigate the relationship between inherited differences in sensitivity to stress and proneness to an increased growth rate of subcutaneously inoculated melanoma. These lines display several genetic and physiological differences, among which distinct sensitivity to mutagens and susceptibility to cancer are especially noticeable. High analgesic mice display high proneness both to stress and a rapid local spread of B16F0 melanoma. However, stress-resistant LA mice do not develop melanoma tumors after inoculation, or if so, tumors regress spontaneously. We found that the chronic mild stress (CMS) procedure leads to enhanced interlinear differences in melanoma susceptibility. Tumors developed faster in stress conditions in both lines. However, LA mice still displayed a tendency for spontaneous regression, and 50% of LA mice did not develop a tumor, even under stressed conditions. Moreover, we showed that chronic stress, but not tumor progression, induces depressive behavior, which may be an important clue in cancer therapy. Our results clearly indicate how the interaction between genetic susceptibility to stress and environmental stress determine the risk and progression of melanoma. To our knowledge, HA/LA mouse lines are the first animal models of distinct melanoma progression mediated by inherited differences in stress reactivity.

  7. Tumor-line specific causes of intertumor heterogeneity in blood supply in human melanoma xenografts.

    PubMed

    Simonsen, Trude G; Gaustad, Jon-Vidar; Leinaas, Marit N; Rofstad, Einar K

    2013-01-01

    The efficacy of most cancer treatments is strongly influenced by the tumor blood supply. The results of experimental studies using xenografted tumors to evaluate novel cancer treatments may therefore vary considerably depending on the blood supply of the specific tumor model being used. Mechanisms underlying intertumor heterogeneity in the blood supply of xenografted tumors derived from same tumor line are poorly understood, and were investigated here by using intravital microscopy to assess tumor blood supply and vascular morphology in human melanomas growing in dorsal window chambers in BALB/c nu/nu mice. Two melanoma lines, A-07 and R-18, were included in the study. These lines differed substantially in angiogenic profiles. Thus, when the expression of 84 angiogenesis-related genes was investigated with a quantitative PCR array, 25% of these genes showed more than a 10-fold difference in expression. Furthermore, A-07 tumors showed higher vascular density, higher vessel tortuosity, higher vessel diameters, shorter vessel segments, and more chaotic vascular architecture than R-18 tumors. Both lines showed large intertumor heterogeneity in blood supply. In the A-07 line, tumors with low microvascular density, long vessel segment, and high vessel tortuosity showed poor blood supply, whereas in the R-18 line, poor tumor blood supply was associated with low tumor arteriolar diameters. Thus, tumor-line specific causes of intertumor heterogeneity in blood supply were identified in human melanoma xenografts, and these tumor-line specific mechanisms were possibly a result of tumor-line specific angiogenic profiles. Copyright © 2012 Elsevier Inc. All rights reserved.

  8. Efficient TGF-β/SMAD signaling in human melanoma cells associated with high c-SKI/SnoN expression

    PubMed Central

    2011-01-01

    Background SKI and SnoN proteins have been shown to inhibit TGF-β signaling, acting both as transcriptional co-repressors in the cell nucleus, and as sequestrators of SMAD proteins in the cytoplasm. TGF-β, on the other hand, induces rapid, proteasome-mediated, degradation of both proteins. How elevated SKI and SnoN protein levels co-exist with active autocrine TGF-β signaling in cancer cells is yet to be understood. Results In this study, we found elevated SKI and SnoN protein levels in a panel of melanoma cell lines, as compared to normal melanocytes. There was no correlation between SKI protein content and the capacity of melanoma cells to invade Matrigel™, to form subcutaneous tumors, or to metastasize to bone after intracardiac inoculation into nude mice. Nor did we find a correlation between SKI expression and histopathological staging of human melanoma. TGF-β induced a rapid and dose-dependent degradation of SKI protein, associated with SMAD3/4 specific transcriptional response and induction of pro-metastatic target genes, partially prevented by pharmacologic blockade of proteasome activity. SKI knockdown in 1205Lu melanoma cells did not alter their invasive capacity or transcriptional responses to TGF-β, and did not allow p21 expression in response to TGF-β or reveal any growth inhibitory activity of TGF-β. Conclusions Despite high expression in melanoma cells, the role of SKI in melanoma remains elusive: SKI does not efficiently interfere with the pro-oncogenic activities of TGF-β, unless stabilized by proteasome blockade. Its highly labile nature makes it an unlikely target for therapeutic intervention. PMID:21211030

  9. Identification of progenitor cancer stem cell in lentigo maligna melanoma.

    PubMed

    Bongiorno, M R; Doukaki, S; Malleo, F; Aricò, M

    2008-07-01

    The potential role of stem cells in neoplasia has aroused considerable interest over the past few years. A number of known biologic characteristics of melanomas support the theory that they may originate in a mutated stem cell. Melanocytic stem cell markers have been described recently. Moreover, the CD133 cells that show surface markers for CD34 are stem cells primitive. These stem cells are capable of differentiating into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. The identification of cancer stem/initiating cells with a crucial role in tumor formation may open up new pharmacologic perspectives. The purpose of this study is to detect the expression of CD133 and CD34, two putative markers of cancer stem cells in the lentigo maligna melanoma. Thirty cases of lentigo maligna melanoma were analyzed using indirect immunohistochemical staining. The vast majority of the samples analyzed showed the presence of rare cells, which were clearly positive for CD133 and CD34. Strong CD133 and CD34 staining was found in the outer root sheath of the mid-lower hair follicles, intermixed with atypical melanocytes extending along layers of the hair follicles. A number of these staminal cells were adjacent and intermixed with melanoma cells. This study supports the stem cell origin of this tumor and suggests that the precursor of the melanoma in question is a stem-like cell rather than the primitive melanoblast committed to be exclusively involved in melanocytic differentiation.

  10. Systematic screening of isogenic cancer cells identifies DUSP6 as context-specific synthetic lethal target in melanoma

    PubMed Central

    Wittig-Blaich, Stephanie; Wittig, Rainer; Schmidt, Steffen; Lyer, Stefan; Bewerunge-Hudler, Melanie; Gronert-Sum, Sabine; Strobel-Freidekind, Olga; Müller, Carolin; List, Markus; Jaskot, Aleksandra; Christiansen, Helle; Hafner, Mathias; Schadendorf, Dirk; Block, Ines; Mollenhauer, Jan

    2017-01-01

    Next-generation sequencing has dramatically increased genome-wide profiling options and conceptually initiates the possibility for personalized cancer therapy. State-of-the-art sequencing studies yield large candidate gene sets comprising dozens or hundreds of mutated genes. However, few technologies are available for the systematic downstream evaluation of these results to identify novel starting points of future cancer therapies. We improved and extended a site-specific recombination-based system for systematic analysis of the individual functions of a large number of candidate genes. This was facilitated by a novel system for the construction of isogenic constitutive and inducible gain- and loss-of-function cell lines. Additionally, we demonstrate the construction of isogenic cell lines with combinations of the traits for advanced functional in vitro analyses. In a proof-of-concept experiment, a library of 108 isogenic melanoma cell lines was constructed and 8 genes were identified that significantly reduced viability in a discovery screen and in an independent validation screen. Here, we demonstrate the broad applicability of this recombination-based method and we proved its potential to identify new drug targets via the identification of the tumor suppressor DUSP6 as potential synthetic lethal target in melanoma cell lines with BRAF V600E mutations and high DUSP6 expression. PMID:28423600

  11. Photoacoustic imaging of single circulating melanoma cells in vivo

    NASA Astrophysics Data System (ADS)

    Wang, Lidai; Yao, Junjie; Zhang, Ruiying; Xu, Song; Li, Guo; Zou, Jun; Wang, Lihong V.

    2015-03-01

    Melanoma, one of the most common types of skin cancer, has a high mortality rate, mainly due to a high propensity for tumor metastasis. The presence of circulating tumor cells (CTCs) is a potential predictor for metastasis. Label-free imaging of single circulating melanoma cells in vivo provides rich information on tumor progress. Here we present photoacoustic microscopy of single melanoma cells in living animals. We used a fast-scanning optical-resolution photoacoustic microscope to image the microvasculature in mouse ears. The imaging system has sub-cellular spatial resolution and works in reflection mode. A fast-scanning mirror allows the system to acquire fast volumetric images over a large field of view. A 500-kHz pulsed laser was used to image blood and CTCs. Single circulating melanoma cells were imaged in both capillaries and trunk vessels in living animals. These high-resolution images may be used in early detection of CTCs with potentially high sensitivity. In addition, this technique enables in vivo study of tumor cell extravasation from a primary tumor, which addresses an urgent pre-clinical need.

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

    PubMed

    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.

  13. The phosphoinositide 3-kinase α selective inhibitor BYL719 enhances the effect of the protein kinase C inhibitor AEB071 in GNAQ/GNA11-mutant uveal melanoma cells.

    PubMed

    Musi, Elgilda; Ambrosini, Grazia; de Stanchina, Elisa; Schwartz, Gary K

    2014-05-01

    G-protein mutations are one of the most common mutations occurring in uveal melanoma activating the protein kinase C (PKC)/mitogen-activated protein kinase and phosphoinositide 3-kinase (PI3K)/AKT pathways. In this study, we described the effect of dual pathway inhibition in uveal melanoma harboring GNAQ and GNA11 mutations via PKC inhibition with AEB071 (sotrastaurin) and PI3K/AKT inhibition with BYL719, a selective PI3Kα inhibitor. Growth inhibition was observed in GNAQ/GNA11-mutant cells with AEB071 versus no activity in wild-type cells. In the GNAQ-mutant cells, AEB071 decreased phosphorylation of myristoylated alanine-rich C-kinase substrate, a substrate of PKC, along with ERK1/2 and ribosomal S6, but persistent AKT activation was present. BYL719 had minimal antiproliferative activity in all uveal melanoma cell lines, and inhibited phosphorylation of AKT in most cell lines. In the GNA11-mutant cell line, similar effects were observed with ERK1/2 inhibition, mostly inhibited by BYL719. With the combination treatment, both GNAQ- and GNA11-mutant cell lines showed synergistic inhibition of cell proliferation and apoptotic cell death. In vivo studies correlated with in vitro findings showing reduced xenograft tumor growth with the combination therapy in a GNAQ-mutant model. These findings suggest a new therapy treatment option for G-protein-mutant uveal melanoma with a focus on specific targeting of multiple downstream pathways as part of combination therapy.

  14. The Phosphoinositide 3-Kinaseα Selective Inhibitor, BYL719, Enhances the Effect of the Protein Kinase C Inhibitor, AEB071, in GNAQ/GNA11 Mutant Uveal Melanoma Cells

    PubMed Central

    Musi, Elgilda; Ambrosini, Grazia; de Stanchina, Elisa; Schwartz, Gary K.

    2014-01-01

    G-protein mutations are one of the most common mutations occurring in uveal melanoma activating the protein kinase C (PKC)/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-Kinase (PI3K)/AKT pathways. In this study, we described the effect of dual pathway inhibition in uveal melanoma harboring GNAQ and GNA11 mutations via PKC inhibition with AEB071 (Sotrastaurin) and PI3k/AKT inhibition with BYL719, a selective PI3Kα inhibitor. Growth inhibition was observed in GNAQ/GNA11 mutant cells with AEB071 versus no activity in WT cells. In the GNAQ-mutant cells, AEB071 decreased phosphorylation of MARCKS, a substrate of PKC, along with ERK1/2 and ribosomal S6, but persistent AKT activation was present. BYL719 had minimal anti-proliferative activity in all uveal melanoma cell lines, and inhibited phosphorylation of AKT in most cell lines. In the GNA11 mutant cell line, similar effects were observed with ERK1/2 inhibition, mostly inhibited by BYL719. With the combination treatment, both GNAQ and GNA11 mutant cell lines showed synergistic inhibition of cell proliferation and apoptotic cell death. In vivo studies correlated with in vitro findings showing reduced xenograft tumor growth with the combination therapy in a GNAQ mutant model. These findings suggest a new therapy treatment option for G-protein mutant uveal melanoma with a focus on specific targeting of multiple downstream pathways as part of combination therapy. PMID:24563540

  15. Cancer stem cell as therapeutic target for melanoma treatment.

    PubMed

    Alamodi, Abdulhadi A; Eshaq, Abdulaziz M; Hassan, Sofie-Yasmin; Al Hmada, Youssef; El Jamal, Siraj M; Fothan, Ahmed M; Arain, Omair M; Hassan, Sarah-Lilly; Haikel, Youssef; Megahed, Mosaad; Hassan, Mohamed

    2016-12-01

    Human malignant melanoma is a highly aggressive skin tumor that is characterized by its extraordinary heterogeneity, propensity for dissemination to distant organs and resistance to cytotoxic agents. Although chemo- and immune-based therapies have been evaluated in clinical trials, most of these therapeutics do not show significant benefit for patients with advanced disease. Treatment failure in melanoma patients is attributed mainly to the development of tumor heterogeneity resulting from the formation of genetically divergent subpopulations. These subpopulations are composed of cancer stem-like cells (CSCs) as a small fraction and non-cancer stem cells that form the majority of the tumor mass. In recent years, CSCs gained more attention and suggested as valuable experimental model system for tumor study. In melanoma, intratumoral heterogeneity, progression and drug resistance result from the unique characteristics of melanoma stem cells (MSCs). These MSCs are characterized by their distinct protein signature and tumor growth-driving pathways, whose activation is mediated by driver mutation-dependent signal. The molecular features of MSCs are either in a causal or consequential relationship to melanoma progression, drug resistance and relapse. Here, we review the current scientific evidence that supports CSC hypothesis and the validity of MSCs-dependent pathways and their key molecules as potential therapeutic target for melanoma treatment.

  16. Mechanisms of Tanshinone II a inhibits malignant melanoma development through blocking autophagy signal transduction in A375 cell.

    PubMed

    Li, Xiaojing; Li, Zhifeng; Li, Xianping; Liu, Baoguo; Liu, Zhijun

    2017-05-22

    Malignant melanoma (MM) is one of the high degree of malignancy and early prone to blood and lymph node metastasis. There is not cured for MM. Tan II A has been reported to reduce cancer cell proliferation. But the mechanism by which Tan II A inhibited melanoma growth are not well characterized. We sought to explore the possible mechanism by which Tan II A regulated cell proliferation through autophagy signaling pathway in A375 cells. We tested the effects of Tan II A on melanoma A375, MV3, M14, and other human cell lines including Hacat and HUVEC cells in cell culture model. Cell proliferation was assessed by using methyl thiazol tetrazolium (MTT) assay. Cell migration ability melanoma A375 was monitored by using cell scratch assay. Transwell chamber experimental was performed to assess the effect of Tan II A on A375 melanoma cell invasion ability. The autophagy body was examined by using flow cytometry. The expression of autophagy-associated protein beclin-1 and microtubule-associated protein 1 light chain 3(LC3)-II, as well as phosphatidylinositol 3-kinase(PI3K)、protein kinase B (Akt)、mammalian target of rapamycin (mTOR)、p70S6K1 signaling pathways were detected by using Western blotting. The effects of Tan II A on tumor progression was also examined in melanoma A375 induced tumor in mouse model. We found that Tan IIA inhibited melanoma A375, MV3, and M14 cell proliferation in dose and time dependent manner. Tan II A reduced CXCL12-induced A375 cell invasive ability and migration in a dose dependent manner. Tan IIA promoted autophagic body production and increased autophagy-associated protein beclin-1 and LC3-II expression in A375 cells. However, Tan IIA reduced the phosphorylation of PI3K, P-AKT, P-mTOR, and P-p7036k1. We also confirmed that Tan II A reduced melanoma A375 induced tumor volume and weight in mouse model. We concluded that Tan II A reduced A375 cells proliferation by activation of autophagy production, blocked PI3K- Akt - mTOR - p70S6K1

  17. 31P NMR spectroscopy studies of phospholipid metabolism in human melanoma xenograft lines differing in rate of tumour cell proliferation.

    PubMed

    Lyng, H; Olsen, D R; Petersen, S B; Rofstad, E K

    1995-04-01

    The concentration of phospholipid metabolites in tumours has been hypothesized to be related to rate of cell membrane turnover and may reflect rate of cell proliferation. The purpose of the study reported here was to investigate whether 31P NMR resonance ratios involving the phosphomonoester (PME) or phosphodiester (PDE) resonance are correlated to fraction of cells in S-phase or volume-doubling time in experimental tumours. Four human melanoma xenograft lines (BEX-t, HUX-t, SAX-t, WIX-t) were included in the study. The tumours were grown subcutaneously in male BALB/c-nu/nu mice. 31P NMR spectroscopy was performed at a magnetic field strength of 4.7 T. Fraction of cells in S-phase was measured by flow cytometry. Tumour volume-doubling time was determined by Gompertzian analysis of volumetric growth data. BEX-t and SAX-t tumours differed in fraction of cells in S-phase and volume-doubling time, but showed similar 31P NMR resonance ratios. BEX-t and WIX-t tumours showed significantly different 31P NMR resonance ratios but similar fractions of cells in S-phase. The 31P NMR resonance ratios were significantly different for small and large HUX-t tumours even though fraction of cells in S-phase and volume-doubling time did not differ with tumour volume. None of the 31P NMR resonance ratios showed significant increase with increasing fraction of cells in S-phase or significant decrease with increasing tumour volume-doubling time across the four xenograft lines.(ABSTRACT TRUNCATED AT 250 WORDS)

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

    PubMed Central

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

    2014-01-01

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

  19. Histology-specific microRNA alterations in melanoma.

    PubMed

    Poliseno, Laura; Haimovic, Adele; Segura, Miguel F; Hanniford, Douglas; Christos, Paul J; Darvishian, Farbod; Wang, Jinhua; Shapiro, Richard L; Pavlick, Anna C; Berman, Russell S; Hernando, Eva; Zavadil, Jiri; Osman, Iman

    2012-07-01

    We examined the microRNA signature that distinguishes the most common melanoma histological subtypes, superficial spreading melanoma (SSM) and nodular melanoma (NM). We also investigated the mechanisms underlying the differential expression of histology-specific microRNAs. MicroRNA array performed on a training cohort of 82 primary melanoma tumors (26 SSM, 56 NM), and nine congenital nevi (CN) revealed 134 microRNAs differentially expressed between SSM and NM (P<0.05). Out of 134 microRNAs, 126 remained significant after controlling for thickness and 31 were expressed at a lower level in SSM compared with both NM and CN. For seven microRNAs (let-7g, miR-15a, miR-16, miR-138, miR-181a, miR-191, and miR-933), the downregulation was associated with selective genomic loss in SSM cell lines and primary tumors, but not in NM cell lines and primary tumors. The lower expression level of six out of seven microRNAs in SSM compared with NM was confirmed by real-time PCR on a subset of cases in the training cohort and validated in an independent cohort of 97 melanoma cases (38 SSM, 59 NM). Our data support a molecular classification in which SSM and NM are two molecularly distinct phenotypes. Therapeutic strategies that take into account subtype-specific alterations might improve the outcome of melanoma patients.

  20. Histology-Specific MicroRNA Alterations in Melanoma

    PubMed Central

    Poliseno, Laura; Haimovic, Adele; Segura, Miguel F.; Hanniford, Douglas; Christos, Paul J.; Darvishian, Farbod; Wang, Jinhua; Shapiro, Richard L.; Pavlick, Anna C.; Berman, Russell S.; Hernando, Eva; Zavadil, Jiri; Osman, Iman

    2013-01-01

    We examined the microRNA signature that distinguishes the most common melanoma histological subtypes, superficial spreading melanoma (SSM) and nodular melanoma (NM). We also investigated the mechanisms underlying the differential expression of histology-specific microRNAs. MicroRNA array performed on a training cohort of 82 primary melanoma tumors (26 SSM, 56 NM), and nine congenital nevi (CN) revealed 134 microRNAs differentially expressed between SSM and NM (P<0.05). Out of 134 microRNAs, 126 remained significant after controlling for thickness and 31 were expressed at a lower level in SSM compared with both NM and CN. For seven microRNAs (let-7g, miR-15a, miR-16, miR-138, miR-181a, miR-191, and miR-933), the downregulation was associated with selective genomic loss in SSM cell lines and primary tumors, but not in NM cell lines and primary tumors. The lower expression level of six out of seven microRNAs in SSM compared with NM was confirmed by real-time PCR on a subset of cases in the training cohort and validated in an independent cohort of 97 melanoma cases (38 SSM, 59 NM). Our data support a molecular classification in which SSM and NM are two molecularly distinct phenotypes. Therapeutic strategies that take into account subtype-specific alterations might improve the outcome of melanoma patients. PMID:22551973

  1. Targeting the hedgehog transcription factors GLI1 and GLI2 restores sensitivity to vemurafenib-resistant human melanoma cells

    PubMed Central

    Faião-Flores, F; Alves-Fernandes, D K; Pennacchi, P C; Sandri, S; Vicente, A L S A; Scapulatempo-Neto, C; Vazquez, V L; Reis, R M; Chauhan, J; Goding, C R; Smalley, K S; Maria-Engler, S S

    2017-01-01

    BRAF inhibitor (BRAFi) therapy for melanoma patients harboring the V600E mutation is initially highly effective, but almost all patients relapse within a few months. Understanding the molecular mechanisms underpinning BRAFi-based therapy is therefore an important issue. Here we identified a previously unsuspected mechanism of BRAFi resistance driven by elevated Hedgehog (Hh) pathway activation that is observed in a cohort of melanoma patients after vemurafenib treatment. Specifically, we demonstrate that melanoma cell lines, with acquired in vitro-induced vemurafenib resistance, show increased levels of glioma-associated oncogene homolog 1 and 2 (GLI1/GLI2) compared with naïve cells. We also observed these findings in clinical melanoma specimens. Moreover, the increased expression of the transcription factors GLI1/GLI2 was independent of canonical Hh signaling and was instead correlated with the noncanonical Hh pathway, involving TGFβ/SMAD (transforming growth factor-β/Sma- and Mad-related family) signaling. Knockdown of GLI1 and GLI2 restored sensitivity to vemurafenib-resistant cells, an effect associated with both growth arrest and senescence. Treatment of vemurafenib-resistant cells with the GLI1/GLI2 inhibitor Gant61 led to decreased invasion of the melanoma cells in a three-dimensional skin reconstruct model and was associated with a decrease in metalloproteinase (MMP2/MMP9) expression and microphthalmia transcription factor upregulation. Gant61 monotherapy did not alter the drug sensitivity of naïve cells, but could reverse the resistance of melanoma cells chronically treated with vemurafenib. We further noted that alternating dosing schedules of Gant61 and vemurafenib prevented the onset of BRAFi resistance, suggesting that this could be a potential therapeutic strategy for the prevention of therapeutic escape. Our results suggest that targeting the Hh pathway in BRAFi-resistant melanoma may represent a viable therapeutic strategy to restore vemurafenib

  2. Targeting the hedgehog transcription factors GLI1 and GLI2 restores sensitivity to vemurafenib-resistant human melanoma cells.

    PubMed

    Faião-Flores, F; Alves-Fernandes, D K; Pennacchi, P C; Sandri, S; Vicente, A L S A; Scapulatempo-Neto, C; Vazquez, V L; Reis, R M; Chauhan, J; Goding, C R; Smalley, K S; Maria-Engler, S S

    2017-03-30

    BRAF inhibitor (BRAFi) therapy for melanoma patients harboring the V600E mutation is initially highly effective, but almost all patients relapse within a few months. Understanding the molecular mechanisms underpinning BRAFi-based therapy is therefore an important issue. Here we identified a previously unsuspected mechanism of BRAFi resistance driven by elevated Hedgehog (Hh) pathway activation that is observed in a cohort of melanoma patients after vemurafenib treatment. Specifically, we demonstrate that melanoma cell lines, with acquired in vitro-induced vemurafenib resistance, show increased levels of glioma-associated oncogene homolog 1 and 2 (GLI1/GLI2) compared with naïve cells. We also observed these findings in clinical melanoma specimens. Moreover, the increased expression of the transcription factors GLI1/GLI2 was independent of canonical Hh signaling and was instead correlated with the noncanonical Hh pathway, involving TGFβ/SMAD (transforming growth factor-β/Sma- and Mad-related family) signaling. Knockdown of GLI1 and GLI2 restored sensitivity to vemurafenib-resistant cells, an effect associated with both growth arrest and senescence. Treatment of vemurafenib-resistant cells with the GLI1/GLI2 inhibitor Gant61 led to decreased invasion of the melanoma cells in a three-dimensional skin reconstruct model and was associated with a decrease in metalloproteinase (MMP2/MMP9) expression and microphthalmia transcription factor upregulation. Gant61 monotherapy did not alter the drug sensitivity of naïve cells, but could reverse the resistance of melanoma cells chronically treated with vemurafenib. We further noted that alternating dosing schedules of Gant61 and vemurafenib prevented the onset of BRAFi resistance, suggesting that this could be a potential therapeutic strategy for the prevention of therapeutic escape. Our results suggest that targeting the Hh pathway in BRAFi-resistant melanoma may represent a viable therapeutic strategy to restore vemurafenib

  3. Epigenetic regulation of REG1A and chemosensitivity of cutaneous melanoma

    PubMed Central

    Sato, Yusuke; Marzese, Diego M; Ohta, Katsuya; Huang, Sharon K; Sim, Myung Shin; Chong, Kelly; Hoon, Dave SB

    2013-01-01

    Regenerating gene 1A (REG1A) plays an important role in tissue regeneration and in cell proliferation in epithelium origin tumors; however, its role in melanoma has not been explored in details. The objective of this study was to identify whether REG1A is expressed in cutaneous melanoma and if REG1A expression status can predict prognosis in cutaneous melanoma patients with metastasis. We also determined whether epigenetic regulation of the promoter region regulates REG1A expression. AJCC stage III cutaneous melanoma specimens with clinically well annotated stage III lymph node melanoma metastasis tissue microarray were assessed by IHC. MALDI-TOF-mass spectrometry and HM450K array were used to identify REG1A promoter region CpG site methylation. Chemotherapeutic agent response by melanoma cells as related to REG1A protein expression was assessed. Post-surgery melanoma patients followed by adjuvant chemotherapy with high REG1A expression had a significantly better prognosis (disease-specific survival) compared with patients with low REG1A expression (log rank test; p = 0.0013). The demethylating reagent 5-Aza-2′-deoxycytidine activated REG1A promoter region resulting in enhanced REG1A mRNA and protein expression in melanoma cell lines. Promoter region CpG methylation was shown to regulate REG1A expression in melanoma cells. Moreover, melanoma lines with high REG1A mRNA expression were more susceptible to Dacarbazine and Cisplatin, as compared with those with low REG1A mRNA expression. In conclusion, REG1A expression status may be useful as a biomarker in melanoma patients for sensitivity to these chemotherapeutic agents. The epigenetic regulation of the REG1A promoter region may offer a potential therapeutic approach to improve chemotherapy for metastatic melanoma patients. PMID:23903855

  4. Loss of cell invasiveness through PKC-mediated syndecan-1 downregulation in melanoma cells under anchorage independency.

    PubMed

    Wang, ChiaChen; Tseng, TingTing; Jhang, Yaoyun; Tseng, JenChih; Hsieh, ChiaoHui; Wu, Wen-guey; Lee, ShaoChen

    2014-11-01

    Anchorage-independent survival is one of the key features for malignant tumor cells. Whether specific gene alterations contributed by anchorage independency would further affect metastatic phenotypes of melanoma cells was unclear. We adapted suspension culture of melanoma cells to establish anchorage independency. The suspended melanoma cells lost their invasive abilities in vitro. Specific loss of laminin-binding ability in suspended melanoma cells was observed, which was correlated with downregulation of syndecan-1 as revealed by microarray and validated by qPCR and Western blot. Modulation of syndecan-1 expression level affected laminin binding, transwell migration and matrix metalloproteinase-2 secretion in melanoma cells. SDC1 expression and transwell migration were correlated with activity or level of protein kinase Cδ as evidence by specific inhibitors and shRNA transfection. In this study, we compared metastatic phenotypes and gene expressions of adherent and suspended melanoma cells. The anchorage independency led to protein kinase Cδ-mediated syndecan-1 downregulation, which contributed to loss of laminin-binding ability, reduced metalloproteinase-2 secretion and loss of invasiveness. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  5. Overexpression of connexin 43 reduces melanoma proliferative and metastatic capacity

    PubMed Central

    Tittarelli, A; Guerrero, I; Tempio, F; Gleisner, M A; Avalos, I; Sabanegh, S; Ortíz, C; Michea, L; López, M N; Mendoza-Naranjo, A; Salazar-Onfray, F

    2015-01-01

    Background: Alterations in connexin 43 (Cx43) expression and/or gap junction (GJ)-mediated intercellular communication are implicated in cancer pathogenesis. Herein, we have investigated the role of Cx43 in melanoma cell proliferation and apoptosis sensitivity in vitro, as well as metastatic capability and tumour growth in vivo. Methods: Connexin 43 expression levels, GJ coupling and proliferation rates were analysed in four different human melanoma cell lines. Furthermore, tumour growth and lung metastasis of high compared with low Cx43-expressing FMS cells were evaluated in vivo using a melanoma xenograft model. Results: Specific inhibition of Cx43 channel activity accelerated melanoma cell proliferation, whereas overexpression of Cx43 increased GJ coupling and reduced cell growth. Moreover, Cx43 overexpression in FMS cells increased basal and tumour necrosis factor-α-induced apoptosis and resulted in decreased melanoma tumour growth and lower number and size of metastatic foci in vivo. Conclusions: Our findings reveal an important role for Cx43 in intrinsically controlling melanoma growth, death and metastasis, and emphasise the potential use of compounds that selectively enhance Cx43 expression on melanoma in the future chemotherapy and/or immunotherapy protocols. PMID:26135897

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

    PubMed

    Vasconcellos, Marne C; Bezerra, Daniel P; Fonseca, Aluísio M; Araújo, Ana Jérsia; Pessoa, Cláudia; Lemos, Telma L G; Costa-Lotufo, Letícia V; de Moraes, Manoel Odorico; Montenegro, Raquel C

    2011-04-01

    Biflorin, an ortho-naphthoquinone, is an active compound found in the roots of Capraria biflora L. It has been reported that biflorin presents anticancer activity, inhibiting both tumor cell line growth in culture and tumor development in mice. The aim of this study was to examine the effectiveness of biflorin treatment using both in-vitro and in-vivo melanoma models. Biflorin displayed considerable cytotoxicity against all tested cell lines, with half maximal inhibitory concentration values ranging from 0.58 μg/ml in NCI H23 (human lung adenocarcinoma) to 14.61 μg/ml in MDA-MB-231 (human breast cancer) cell lines. In a second set of experiments using B16 melanoma cells as a model, biflorin reduced cell viability but did not cause significant increase in the number of nonviable cells. In addition, the DNA synthesis was significantly inhibited. Flow cytometry analysis showed that biflorin may lead to an apoptotic death in melanoma cells, inducing DNA fragmentation and mitochondria depolarization, without affecting membrane integrity. In B16 melanoma-bearing mice, administration of biflorin (25mg/day) for 10 days inhibited tumor growth, and also increased the mean survival rate from 33.3±0.9 days (control) to 44.5±3.4 days (treated). Our findings suggest that biflorin may be considered as a promising lead compound for designing new drugs to be used in the treatment of melanoma.

  7. SOX2 and nestin expression in human melanoma: an immunohistochemical and experimental study

    PubMed Central

    Laga, Alvaro C.; Zhan, Qian; Weishaupt, Carsten; Ma, Jie; Frank, Markus H.; Murphy, George F.

    2012-01-01

    SOX2 is an embryonic neural crest stem-cell transcription factor recently shown to be expressed in human melanoma and to correlate with experimental tumor growth. SOX2 binds to an enhancer region of the gene that encodes for nestin, also a neural progenitor cell biomarker. To define further the potential relationship between SOX2 and nestin, we examined co-expression patterns in 135 melanomas and 37 melanocytic nevi. Immunohistochemical staining in 27 melanoma tissue sections showed an association between SOX2 positivity, spindle cell shape and a peripheral nestin distribution pattern. In contrast, SOX2-negative cells were predominantly epithelioid, and exhibited a cytoplasmic pattern for nestin. In tissue microarrays, co-expression correlated with tumor progression, with only 11% of nevi co-expressing SOX2 and nestin in contrast to 65% of metastatic melanomas, and preliminarily, with clinical outcome. Human melanoma lines that differentially expressed constitutive SOX2 revealed a positive correlation between SOX2 and nestin expression. Experimental melanomas grown from these respective cell lines in murine subcutis and dermis of xenografted human skin maintained the association between SOX2-positivity, spindle cell shape, and peripheral nestin distribution. Moreover, the cytoplasmic pattern of nestin distribution was observed in xenografts generated from SOX2-knockdown A2058 melanoma cells, in contrast to the periperhal nestin pattern seen in tumors grown from A2058 control cells transfected with non-target shRNA. In aggregate, these data further support a biologically significant linkage between SOX2 and nestin expression in human melanoma. PMID:21410764

  8. Bis-anthracycline WP760 abrogates melanoma cell growth by transcription inhibition, p53 activation and IGF1R downregulation.

    PubMed

    Olbryt, Magdalena; Rusin, Aleksandra; Fokt, Izabela; Habryka, Anna; Tudrej, Patrycja; Student, Sebastian; Sochanik, Aleksander; Zieliński, Rafał; Priebe, Waldemar

    2017-10-01

    Anthracycline chemotherapeutics, e.g. doxorubicin and daunorubicin, are active against a broad spectrum of cancers. Their cytotoxicity is mainly attributed to DNA intercalation, interference with topoisomerase activity, and induction of double-stranded DNA breaks. Since modification of anthracyclines can profoundly affect their pharmacological properties we attempted to elucidate the mechanism of action, and identify possible molecular targets, of bis-anthracycline WP760 which previously demonstrated anti-melanoma activity at low nanomolar concentrations. We studied the effect of WP760 on several human melanoma cell lines derived from tumors in various development stages and having different genetic backgrounds. WP760 inhibited cell proliferation (IC 50  = 1-99 nM), impaired clonogenic cell survival (100 nM), and inhibited spheroid growth (≥300 nM). WP760 did not induce double-stranded DNA breaks but strongly inhibited global transcription. Moreover, WP760 caused nucleolar stress and led to activation of the p53 pathway. PCR array analysis showed that WP760 suppressed transcription of ten genes (ABCC1, MTOR, IGF1R, EGFR, GRB2, PRKCA, PRKCE, HDAC4, TXNRD1, AKT1) associated with, inter alia, cytoprotective mechanisms initiated in cancer cells during chemotherapy. Furthermore, WP760 downregulated IGF1R and upregulated PLK2 expression in most of the tested melanoma cell lines. These results suggest that WP760 exerts anti-melanoma activity by targeting global transcription and activation of the p53 pathway and could become suitable as an effective therapeutic agent.

  9. Pleckstrin homology domain-interacting protein (PHIP) as a marker and mediator of melanoma metastasis

    PubMed Central

    De Semir, David; Nosrati, Mehdi; Bezrookove, Vladimir; Dar, Altaf A.; Federman, Scot; Bienvenu, Geraldine; Venna, Suraj; Rangel, Javier; Climent, Joan; Meyer Tamgüney, Tanja M.; Thummala, Suresh; Tong, Schuyler; Leong, Stanley P. L.; Haqq, Chris; Billings, Paul; Miller, James R.; Sagebiel, Richard W.; Debs, Robert; Kashani-Sabet, Mohammed

    2012-01-01

    Although melanomas with mutant v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) can now be effectively targeted, there is no molecular target for most melanomas expressing wild-type BRAF. Here, we show that the activation of Pleckstrin homology domain-interacting protein (PHIP), promotes melanoma metastasis, can be used to classify a subset of primary melanomas, and is a prognostic biomarker for melanoma. Systemic, plasmid-based shRNA targeting of Phip inhibited the metastatic progression of melanoma, whereas stable suppression of Phip in melanoma cell lines suppressed metastatic potential and prolonged the survival of tumor-bearing mice. The human PHIP gene resides on 6q14.1, and although 6q loss has been observed in melanoma, the PHIP locus was preserved in melanoma cell lines and patient samples, and its overexpression was an independent adverse predictor of survival in melanoma patients. In addition, a high proportion of PHIP-overexpressing melanomas harbored increased PHIP copy number. PHIP-overexpressing melanomas include tumors with wild-type BRAF, neuroblastoma RAS viral (v-ras) oncogene homolog, and phosphatase and tensin homolog, demonstrating PHIP activation in triple-negative melanoma. These results describe previously unreported roles for PHIP in predicting and promoting melanoma metastasis, and in the molecular classification of melanoma. PMID:22511720

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

    PubMed Central

    Ivanov, Vladimir N.; Hei, Tom K.

    2015-01-01

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

  11. ABCB1 identifies a subpopulation of uveal melanoma cells with high metastatic propensity

    PubMed Central

    Landreville, Solange; Agapova, Olga A.; Kneass, Zachary T.; Salesse, Christian; Harbour, J. William

    2011-01-01

    SUMMARY Metastasis of tumor cells to distant organs is the leading cause of death in melanoma. Yet, the mechanisms of metastasis remain poorly understood. One key question is whether all cells in a primary tumor are equally likely to metastasize or whether subpopulations of cells preferentially give rise to metastases. Here, we identified a subpopulation of uveal melanoma cells expressing the multidrug resistance transporter ABCB1 that are highly metastatic compared to ABCB1− bulk tumor cells. ABCB1+ cells also exhibited enhanced clonogenicity, anchorage independent growth, tumorigenicity and mitochondrial activity compared to ABCB1− cells. A375 cutaneous melanoma cells contained a similar subpopulation of highly metastatic ABCB1+ cells. These findings suggest that some uveal melanoma cells have greater potential for metastasis than others, and that a better understanding of such cells may be necessary for more successful therapies for metastatic melanoma. PMID:21575142

  12. MicroRNA signatures differentiate melanoma subtypes

    PubMed Central

    Chan, Elcie; Patel, Rajeshvari; Nallur, Sunitha; Ratner, Elena; Bacchiocchi, Antonella; Hoyt, Kathleen; Szpakowski, Sebastian; Godshalk, Sirie; Ariyan, Stephan; Sznol, Mario; Halaban, Ruth; Krauthammer, Michael; Tuck, David; Slack, Frank J

    2011-01-01

    Melanoma is an aggressive cancer that is highly resistance to therapies once metastasized. We studied microRNA (miRNA) expression in clinical melanoma subtypes and evaluated different miRNA signatures in the background of gain of function somatic and inherited mutations associated with melanoma. Total RNA from 42 patient derived primary melanoma cell lines and three independent normal primary melanocyte cell cultures was evaluated by miRNA array. MiRNA expression was then analyzed comparing subtypes and additional clinicopathologic criteria including somatic mutations. The prevalence and association of an inherited variant in a miRNA binding site in the 3′UTR of the KRAS oncogene, referred to as the KRAS-variant, was also evaluated. We show that seven miRNAs, miR-142-3p, miR-486, miR-214, miR-218, miR-362, miR-650 and miR-31, were significantly correlated with acral as compared to non-acral melanomas (p < 0.04). In addition, we discovered that the KRAS-variant was enriched in non-acral melanoma (25%), and that miR-137 under expression was significantly associated with melanomas with the KRAS-variant. Our findings indicate that miRNAs are differentially expressed in melanoma subtypes and that their misregulation can be impacted by inherited gene variants, supporting the hypothesis that miRNA misregulation reflects biological differences in melanoma. PMID:21543894

  13. Proton beam irradiation inhibits the migration of melanoma cells.

    PubMed

    Jasińska-Konior, Katarzyna; Pochylczuk, Katarzyna; Czajka, Elżbieta; Michalik, Marta; Romanowska-Dixon, Bożena; Swakoń, Jan; Urbańska, Krystyna; Elas, Martyna

    2017-01-01

    In recent years experimental data have indicated that low-energy proton beam radiation might induce a difference in cellular migration in comparison to photons. We therefore set out to compare the effect of proton beam irradiation and X-rays on the survival and long-term migratory properties of two cell lines: uveal melanoma Mel270 and skin melanoma BLM. Cells treated with either proton beam or X-rays were analyzed for their survival using clonogenic assay and MTT test. Long-term migratory properties were assessed with time-lapse monitoring of individual cell movements, wound test and transpore migration, while the expression of the related proteins was measured with western blot. Exposure to proton beam and X-rays led to similar survival but the quality of the cell colonies was markedly different. More paraclones with a low proliferative activity and fewer highly-proliferative holoclones were found after proton beam irradiation in comparison to X-rays. At 20 or 40 days post-irradiation, migratory capacity was decreased more by proton beam than by X-rays. The beta-1-integrin level was decreased in Mel270 cells after both types of radiation, while vimentin, a marker of EMT, was increased in BLM cells only. We conclude that proton beam irradiation induced long-term inhibition of cellular motility, as well as changes in the level of beta-1 integrin and vimentin. If confirmed, the change in the quality, but not in the number of colonies after proton beam irradiation might favor tumor growth inhibition after fractionated proton therapy.

  14. New target genes of MITF-induced microRNA-211 contribute to melanoma cell invasion.

    PubMed

    Margue, Christiane; Philippidou, Demetra; Reinsbach, Susanne E; Schmitt, Martina; Behrmann, Iris; Kreis, Stephanie

    2013-01-01

    The non-coding microRNAs (miRNA) have tissue- and disease-specific expression patterns. They down-regulate target mRNAs, which likely impacts on most fundamental cellular processes. Differential expression patterns of miRNAs are currently being exploited for identification of biomarkers for early disease diagnosis, prediction of progression for melanoma and other cancers and as promising drug targets, since they can easily be inhibited or replaced in a given cellular context. Before successfully manipulating miRNAs in clinical settings, their precise expression levels, endogenous functions and thus their target genes have to be determined. MiR-211, a melanocyte lineage-specific small non-coding miRNA, is located in an intron of TRPM1, a target gene of the microphtalmia-associated transcription factor (MITF). By transcriptionally up-regulating TRPM1, MITF, which is critical for both melanocyte differentiation and survival and for melanoma progression, indirectly drives the expression of miR-211. Expression of this miRNA is often reduced in melanoma samples. Here, we investigated functional roles of miR-211 by identifying and studying new target genes. We show that MITF-correlated miR-211 expression levels are mostly but not always reduced in a panel of 11 melanoma cell lines and in primary and metastatic melanoma compared to normal melanocytes and nevi, respectively. MiR-211 itself only marginally impacted on cell invasion and migration, while perturbation of some new miR-211 target genes, such as AP1S2, SOX11, IGFBP5, and SERINC3 significantly increased invasion. These results and the variable expression levels of miR-211 raise serious doubts on the value of miR-211 as a melanoma tumor-suppressing miRNA and/or as a biomarker for melanoma.

  15. Keratinocytes negatively regulate the N-cadherin levels of melanoma cells via contact-mediated calcium regulation.

    PubMed

    Chung, Heesung; Jung, Hyejung; Jho, Eek-Hoon; Multhaupt, Hinke A B; Couchman, John R; Oh, Eok-Soo

    2018-06-14

    In human skin, melanocytes and their neighboring keratinocytes have a close functional interrelationship. Keratinocytes, which represent the prevalent cell type of human skin, regulate melanocytes through various mechanisms. Here, we use a keratinocyte and melanoma co-culture system to show for the first time that keratinocytes regulate the cell surface expression of N-cadherin through cell-cell contact. Compared to mono-cultured human melanoma A375 cells, which expressed high levels of N-cadherin, those co-cultured with the HaCaT human keratinocyte cell line showed reduced levels of N-cadherin. This reduction was most evident in areas of A375 cells that underwent cell-cell contact with the HaCaT cells, whereas HaCaT cell-derived extracellular matrix and conditioned medium both failed to reduce N-cadherin levels. The intracellular level of calcium in co-cultured A375 cells was lower than that in mono-cultured A375 cells, and treatment with a cell-permeant calcium chelator (BAPTA) reduced the N-cadherin level of mono-cultured A375 cells. Furthermore, co-culture with HaCaT cells reduced the expression levels of transient receptor potential cation channel (TRPC) 1, -3 and -6 in A375 cells, and siRNA-mediated multi-depletion of TRPC1, -3 and -6 reduced the N-cadherin level in these cells. Taken together, these data suggest that keratinocytes negatively regulate the N-cadherin levels of melanoma cells via cell-to-cell contact-mediated calcium regulation. Copyright © 2018. Published by Elsevier Inc.

  16. Exploiting cannabinoid-induced cytotoxic autophagy to drive melanoma cell death.

    PubMed

    Armstrong, Jane L; Hill, David S; McKee, Christopher S; Hernandez-Tiedra, Sonia; Lorente, Mar; Lopez-Valero, Israel; Eleni Anagnostou, Maria; Babatunde, Fiyinfoluwa; Corazzari, Marco; Redfern, Christopher P F; Velasco, Guillermo; Lovat, Penny E

    2015-06-01

    Although the global incidence of cutaneous melanoma is increasing, survival rates for patients with metastatic disease remain <10%. Novel treatment strategies are therefore urgently required, particularly for patients bearing BRAF/NRAS wild-type tumors. Targeting autophagy is a means to promote cancer cell death in chemotherapy-resistant tumors, and the aim of this study was to test the hypothesis that cannabinoids promote autophagy-dependent apoptosis in melanoma. Treatment with Δ(9)-Tetrahydrocannabinol (THC) resulted in the activation of autophagy, loss of cell viability, and activation of apoptosis, whereas cotreatment with chloroquine or knockdown of Atg7, but not Beclin-1 or Ambra1, prevented THC-induced autophagy and cell death in vitro. Administration of Sativex-like (a laboratory preparation comprising equal amounts of THC and cannabidiol (CBD)) to mice bearing BRAF wild-type melanoma xenografts substantially inhibited melanoma viability, proliferation, and tumor growth paralleled by an increase in autophagy and apoptosis compared with standard single-agent temozolomide. Collectively, our findings suggest that THC activates noncanonical autophagy-mediated apoptosis of melanoma cells, suggesting that cytotoxic autophagy induction with Sativex warrants clinical evaluation for metastatic disease.

  17. HDAC6 interacts with PTPN1 to enhance melanoma cells progression.

    PubMed

    Liu, Jiaqi; Luan, Wenjie; Zhang, Yong; Gu, Jianying; Shi, Yuedong; Yang, Yanwen; Feng, Zihao; Qi, Fazhi

    2018-01-22

    Histone deacetylase 6 (HDAC6) plays an important role in oncogenic transformation and cancer metastasis. Our previous study has demonstrated that HDAC6 was highly expressed in melanoma cells, and contributed to the proliferation and metastasis of melanoma cells. However, the underlying mechanism of HDAC6 in melanoma metastasis and progression remains largely unclear. In this study, we reported that HDAC6 directly interacted with Tyrosine-protein phosphatase non-receptor type 1 (PTPN1) by performing co-immunoprecipitation (Co-IP) combined with liquid chromatography tandem mass spectrometry (LC-MS/MS). HDAC6 increased the protein level of PTPN1 independent of histone modifying activity. In addition, PTPN1 promoted proliferation, colony formation and migration while decreased apoptosis of melanoma cells through activating extracellular signal-regulated kinase 1/2 (ERK1/2). Furthermore, we found that matrix metallopeptidase 9 (MMP9) was increased by HDAC6/PTPN1/ERK1/2 axis, which might serve as a mechanism for melanoma invasion and metastasis. In conclusion, HDAC6 might enhance aggressive melanoma cells progression via interacting with PTPN1, which was independent of its histone modifying activity. Copyright © 2017. Published by Elsevier Inc.

  18. Interaction of dacarbazine and imexon, in vitro and in vivo, in human A375 melanoma cells.

    PubMed

    Samulitis, Betty K; Dorr, Robert T; Chow, H-H Sherry

    2011-09-01

    We evaluated mechanisms of interaction between the alkyating agent dacarbazine (DTIC) and the pro-oxidant, imexon, in the human A375 melanoma cell line. The effect of DTIC and imexon, alone and in combination, was evaluated for growth inhibition (MTT), radiolabeled drug uptake, cellular thiol content (HPLC), and DNA strand breaks (Comet assay). Pharmacokinetic and antitumor effects were evaluated in mice. Growth inhibition in vitro was additive with the two drugs. There was no effect on drug uptake or on the number of DNA strand breaks. There was a >75% reduction in cellular glutathione and cysteine with imexon but not DTIC. Co-administration of the two drugs in mice caused an increase in the area under the curve of both drugs, but the combination was not effective in reducing human A375 melanoma tumors in vivo. Imexon and dacarbazine show additive effects in vitro but not in vivo in human A375 melanoma cells.

  19. Interleukin 8 mediates bcl-xL-induced enhancement of human melanoma cell dissemination and angiogenesis in a zebrafish xenograft model.

    PubMed

    Gabellini, Chiara; Gómez-Abenza, Elena; Ibáñez-Molero, Sofia; Tupone, Maria Grazia; Pérez-Oliva, Ana B; de Oliveira, Sofia; Del Bufalo, Donatella; Mulero, Victoriano

    2018-02-01

    The protein bcl-xL is able to enhance the secretion of the proinflammatory chemokine interleukin 8 (CXCL8) in human melanoma lines. In this study, we investigate whether the bcl-xL/CXCL8 axis is important for promoting melanoma angiogenesis and aggressiveness in vivo, using angiogenesis and xenotransplantation assays in zebrafish embryos. When injected into wild-type embryos, bcl-xL-overexpressing melanoma cells showed enhanced dissemination and angiogenic activity compared with control cells. Human CXCL8 protein elicited a strong proangiogenic activity in zebrafish embryos and zebrafish Cxcr2 receptor was identified as the mediator of CXCL8 proangiogenic activity using a morpholino-mediated gene knockdown. However, human CXCL8 failed to induce neutrophil recruitment in contrast to its zebrafish homolog. Interestingly, the greater aggressiveness of bcl-xL-overexpressing melanoma cells was mediated by an autocrine effect of CXCL8 on its CXCR2 receptor, as confirmed by an shRNA approach. Finally, correlation studies of gene expression and survival analyses using microarray and RNA-seq public databases of human melanoma biopsies revealed that bcl-xL expression significantly correlated with the expression of CXCL8 and other markers of melanoma progression. More importantly, a high level of co-expression of bcl-xL and CXCL8 was associated with poor prognosis in melanoma patients. In conclusion, these data demonstrate the existence of an autocrine CXCL8/CXCR2 signaling pathway in the bcl-xL-induced melanoma aggressiveness, encouraging the development of novel therapeutic approaches for high bcl-xL-expressing melanoma. © 2017 UICC.

  20. Bevacizumab plus fotemustine as first-line treatment in metastatic melanoma patients: clinical activity and modulation of angiogenesis and lymphangiogenesis factors.

    PubMed

    Del Vecchio, Michele; Mortarini, Roberta; Canova, Stefania; Di Guardo, Lorenza; Pimpinelli, Nicola; Sertoli, Mario R; Bedognetti, Davide; Queirolo, Paola; Morosini, Paola; Perrone, Tania; Bajetta, Emilio; Anichini, Andrea

    2010-12-01

    To assess the clinical and biological activity of the association of bevacizumab and fotemustine as first-line treatment in advanced melanoma patients. Previously untreated, metastatic melanoma patients (n = 20) received bevacizumab (at 15 mg/kg every 3 weeks) and fotemustine (100 mg/m² by intravenous administration on days 1, 8, and 15, repeated after 4 weeks) in a multicenter, single-arm, open-label, phase II study. Primary endpoint was the best overall response rate; other endpoints were toxicity, time to progression (TTP), and overall survival (OS). Serum cytokines, angiogenesis, and lymphangiogenesis factors were monitored by multiplex arrays and by in vitro angiogenesis assays. Effects of fotemustine on melanoma cells, in vitro, on vascular endothelial growth factor (VEGF)-C release and apoptosis were assessed by ELISA and flow cytometry, respectively. One complete response, 2 partial responses (PR), and 10 patients with stable disease were observed. TTP and OS were 8.3 and 20.5 months, respectively. Fourteen patients experienced adverse events of toxicity grade 3-4. Serum VEGF-A levels in evaluated patients (n = 15) and overall serum proangiogenic activity were significantly inhibited. A significant reduction in VEGF-C levels was found in several post-versus pretherapy serum samples. In vitro, fotemustine inhibited VEGF-C release by melanoma cells without inducing significant cell death. Serum levels of interleukin (IL)-10 and IL-12p70 showed the highest levels in sera of PR patients, compared with patients with stable or progressive disease whereas IL-23 showed the opposite pattern. The combination of bevacizumab plus fotemustine has clinical activity in advanced melanoma and promotes systemic modulation of angiogenesis and lymphangiogenesis factors. ©2010 AACR.

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

    PubMed

    Zhang, Di; Han, Yantao; Xu, Luo

    2016-12-01

    Melanoma is the most malignant type of skin cancer. In recent years, mounting studies have evidenced the involvement of miRNAs in melanoma. One of these miRNAs, miR-124 has been found aberrantly downregulated in a variety of human malignancies. In this study, our results showed that the expression of miR-124 was significantly lower in malignant melanoma tissues and cell lines and miR-124 functioned as a tumor suppressor in melanoma. Moreover, our findings showed that miR-124 exerted anti-tumor effect by directly targeting RLIP76, a stress-inducible non-ABC transporter that plays a crucial role in the development of melanoma. Furthermore, our study also showed that physcion 8-O-β-glucopyranoside, a natural compound from medicinal plant, could inhibit the proliferation and invasion of melanoma cells by targeting miR-124/RLIP76 signaling. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  2. Melanoma Cells Can Adopt the Phenotype of Stromal Fibroblasts and Macrophages by Spontaneous Cell Fusion in Vitro.

    PubMed

    Kemény, Lajos V; Kurgyis, Zsuzsanna; Buknicz, Tünde; Groma, Gergely; Jakab, Ádám; Zänker, Kurt; Dittmar, Thomas; Kemény, Lajos; Németh, István B

    2016-06-02

    After the removal of primary cutaneous melanoma some patients develop local recurrences, even after having histologically tumor-free re-excision. A potential explanation behind this phenomenon is that tumor cells switch their phenotype, making their recognition via standard histopathological assessments extremely difficult. Tumor-stromal cell fusion has been proposed as a potential mechanism for tumor cells to acquire mesenchymal traits; therefore, we hypothesized that melanoma cells could acquire fibroblast- and macrophage-like phenotypes via cell fusion. We show that melanoma cells spontaneously fuse with human dermal fibroblasts and human peripheral blood monocytes in vitro. The hybrid cells' nuclei contain chromosomes from both parental cells and are indistinguishable from the parental fibroblasts or macrophages based on their morphology and immunophenotype, as they could lose the melanoma specific MART1 marker, but express the fibroblast marker smooth muscle actin or the macrophage marker CD68. Our results suggest that, by spontaneous cell fusion in vitro, tumor cells can adopt the morphology and immunophenotype of stromal cells while still carrying oncogenic, tumor-derived genetic information. Therefore, melanoma-stromal cell fusion might play a role in missing tumor cells by routine histopathological assessments.

  3. The Role of BPTF in Melanoma Progression and in Response to BRAF-Targeted Therapy

    PubMed Central

    Dar, Altaf A.; Nosrati, Mehdi; Bezrookove, Vladimir; de Semir, David; Majid, Shahana; Thummala, Suresh; Sun, Vera; Tong, Schuyler; Leong, Stanley P. L.; Minor, David; Billings, Paul R.; Soroceanu, Liliana; Debs, Robert; Miller, James R.; Sagebiel, Richard W.

    2015-01-01

    Background: Bromodomain PHD finger transcription factor (BPTF) plays an important role in chromatin remodeling, but its functional role in tumor progression is incompletely understood. Here we explore the oncogenic effects of BPTF in melanoma. Methods: The consequences of differential expression of BPTF were explored using shRNA-mediated knockdown in several melanoma cell lines. Immunoblotting was used to assess the expression of various proteins regulated by BPTF. The functional role of BPTF in melanoma progression was investigated using assays of colony formation, invasion, cell cycle, sensitivity to selective BRAF inhibitors, and in xenograft models of melanoma progression (n = 12 mice per group). The biomarker role of BPTF in melanoma progression was assessed using fluorescence in situ hybridization and immunohistochemical analyses. All statistical tests were two-sided. Results: shRNA-mediated BPTF silencing suppressed the proliferative capacity (by 65.5%) and metastatic potential (by 66.4%) of melanoma cells. Elevated BPTF copy number (mean ≥ 3) was observed in 28 of 77 (36.4%) melanomas. BPTF overexpression predicted poor survival in a cohort of 311 melanoma patients (distant metastasis-free survival P = .03, and disease-specific survival P = .008), and promoted resistance to BRAF inhibitors in melanoma cell lines. Metastatic melanoma tumors progressing on BRAF inhibitors contained low BPTF-expressing, apoptotic tumor cell subclones, indicating the continued presence of drug-responsive subclones within tumors demonstrating overall resistance to anti-BRAF agents. Conclusions: These studies demonstrate multiple protumorigenic functions for BPTF and identify it as a novel target for anticancer therapy. They also suggest the combination of BPTF targeting with BRAF inhibitors as a novel therapeutic strategy for melanomas with mutant BRAF. PMID:25713167

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

    PubMed Central

    2010-01-01

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

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

    PubMed

    Yu, Ji Sun; Kim, An Keun

    2010-08-24

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

  6. Sodium ascorbate inhibits growth via the induction of cell cycle arrest and apoptosis in human malignant melanoma A375.S2 cells.

    PubMed

    Lin, Shuw-Yuan; Lai, Wan-Wen; Chou, Chi-Chung; Kuo, Hsiu-Maan; Li, Te-Mao; Chung, Jing-Gung; Yang, Jen-Hung

    2006-12-01

    Vitamin C has been reported to be useful in the treatment and prevention of cancer. Inconsistent effects from growth stimulation to induction of apoptosis of malignant tumor cells, however, have been reported. Melanoma is an increasingly common and potentially lethal malignancy. It was reported that melanoma cells were more susceptible to ascorbate toxicity than any other tumor cells. The mechanisms accounting for ascorbate-induced apoptosis in human melanoma cells, however, have remained unclear. This study was undertaken to investigate the effect of sodium ascorbate on cytotoxicity and apoptosis in human malignant melanoma A375.S2 cells. A375.S2 cells were incubated with a certain range of concentrations of sodium ascorbate for various time periods. In order to examine the effects of sodium ascorbate on cell proliferation, cell cycle, apoptosis and necrosis, we performed 4,6-diamidino-2-phenylindole dihydrochloride assays and flow cytometry analysis. Polymerase chain reaction was used to examine the mRNA levels of p53, p21, p27, cyclin A, cyclin E, CDK2 and CDK4, which are associated with cell cycle S-phase arrest and apoptosis. Flow cytometric analysis showed that sodium ascorbate significantly induced cell cycle arrest and apoptosis in the A375.S2 cell line in a dose-dependent manner. The increased expressions of p53 and p21, and the decreased expressions of cyclin A, cyclin E, CDK2 and CDK4, indicated the cell cycle arrest at G1/S phase after the cells had been treated with sodium ascorbate. Induction of apoptosis involved an increase in the levels of p53, p21 and cellular Ca, and a decrease in mitochondrial membrane potential and activation of caspase 3 before culminating in apoptosis in sodium ascorbate-treated A375.S2 cells.

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

    PubMed

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

    2018-01-01

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

  8. FOXP3 expression is modulated by TGF-β1/NOTCH1 pathway in human melanoma

    PubMed Central

    Skarmoutsou, Eva; Bevelacqua, Valentina; D'Amico, Fabio; Russo, Angela; Spandidos, Demetrios A.; Scalisi, Aurora

    2018-01-01

    Forkhead box protein 3 (FOXP3) transcription factor is expressed by immune cells and several human cancers and is associated with tumor aggressiveness and unfavorable clinical outcomes. NOTCH and transforming growth factor-β (TGF-β) protumorigenic effects are mediated by FOXP3 expression in several cancer models; however, their interaction and role in melanoma is unknown. We investigated TGF-β-induced FOXP3 gene expression during NOTCH1 signaling inactivation. Primary (WM35) and metastatic melanoma (A375 and A2058) cell lines and normal melanocytes (NHEM) were used. FOXP3 subcellular distribution was evaluated by immuno cytochemical analysis. Gene expression levels were assessed by reverse transcription-quantitative polymerase chain reaction. Protein levels were assessed by western blot analysis. The γ-secretase inhibitor (GSI) was used for NOTCH1 inhibition and recombinant human (rh)TGF-β was used for melanoma cell stimulation. Cell proliferation and viability were respectively assessed by MTT and Trypan blue dye assays. FOXP3 mRNA and protein levels were progressively higher in WM35, A375 and A2058 cell lines compared to NHEM and their levels were further increased after stimulation with rh-TGF-β. TGF-β-mediated FOXP3 expression was mediated by NOTCH1 signaling. Inhibition of NOTCH1 with concomitant rh-TGF-β stimulation determined the reduction in gene expression and protein level of FOXP3. Finally, melanoma cell line proliferation and viability were reduced by NOTCH1 inhibition. The results show that nn increase in FOXP3 expression in metastatic melanoma cell lines is a potential marker of tumor aggressiveness and metastasis. NOTCH1 is a central mediator of TGF-β-mediated FOXP3 expression and NOTCH1 inhibition produces a significant reduction of melanoma cell proliferation and viability. PMID:29620159

  9. Cytologic Features of Malignant Melanoma with Osteoclast-Like Giant Cells.

    PubMed

    Jiménez-Heffernan, José A; Adrados, Magdalena; Muñoz-Hernández, Patricia; Fernández-Rico, Paloma; Ballesteros-García, Ana I; Fraga, Javier

    2018-01-01

    Malignant melanoma showing numerous osteoclast-like giant cells (OGCs) is an uncommon morphologic phenomenon, rarely mentioned in the cytologic literature. The few reported cases seem to have an aggressive clinical behavior. Although most findings support monocyte/macrophage differentiation, the exact nature of OGCs is not clear. A 57-year-old woman presented with an inguinal lymphadenopathy. Sixteen years before, cutaneous malignant melanoma of the lower limb had been excised. Needle aspiration revealed abundant neoplastic single cells as well as numerous multinucleated OGCs. Occasional neoplastic giant cells were also present. Nuclei of OGCs were monomorphic with oval morphology and were smaller than those of melanoma cells. The immunophenotype of OGCs (S100-, HMB45-, Melan-A-, SOX10-, Ki67-, CD163-, BRAF-, CD68+, MiTF+, p16+) was the expected for reactive OGCs of monocyte/macrophage origin. The tumor has shown an aggressive behavior with further metastases to the axillary lymph nodes and oral cavity. Numerous OGCs are a rare and relevant finding in malignant melanoma. Their presence should not induce confusion with other tumors rich in osteoclastic cells. Since a relevant number of OGCs in melanoma may mean a more aggressive behavior, and patients may benefit from specific treatments, their presence should be mentioned in the pathologic report. © 2018 S. Karger AG, Basel.

  10. Basal cell carcinoma, squamous cell carcinoma and melanoma of the head and face.

    PubMed

    Feller, L; Khammissa, R A G; Kramer, B; Altini, M; Lemmer, J

    2016-02-05

    Ultraviolet light (UV) is an important risk factor for cutaneous basal cell carcinoma, cutaneous squamous cell carcinoma and cutaneous melanoma of the skin. These cancers most commonly affect persons with fair skin and blue eyes who sunburn rather than suntan. However, each of these cancers appears to be associated with a different pattern of UV exposure and to be mediated by different intracellular molecular pathways.Some melanocortin 1 receptor (MC1R) gene variants play a direct role in the pathogenesis of cutaneous basal cell carcinoma, cutaneous squamous cell carcinoma and cutaneous melanoma apart from their role in determining a cancer-prone pigmentory phenotype (fair skin, red hair, blue eyes) through their interactions with other genes regulating immuno-inflammatory responses, DNA repair or apoptosis.In this short review we focus on the aetiological role of UV in cutaneous basal cell carcinoma, cutaneous squamous cell carcinoma and cutaneous melanoma of the skin, and on some associated biopathological events.

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

    PubMed Central

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

    2017-01-01

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

  12. Odontogenic ameloblast-associated protein (ODAM) inhibits growth and migration of human melanoma cells and elicits PTEN elevation and inactivation of PI3K/AKT signaling

    PubMed Central

    2013-01-01

    Background The Odontogenic Ameloblast-associated Protein (ODAM) is expressed in a wide range of normal epithelial, and neoplastic tissues, and we have posited that ODAM serves as a novel prognostic biomarker for breast cancer and melanoma. Transfection of ODAM into breast cancer cells yields suppression of cellular growth, motility, and in vivo tumorigenicity. Herein we have extended these studies to the effects of ODAM on cultured melanoma cell lines. Methods The A375 and C8161 melanoma cell lines were stably transfected with ODAM and assayed for properties associated with tumorigenicity including cell growth, motility, and extracellular matrix adhesion. In addition, ODAM–transfected cells were assayed for signal transduction via AKT which promotes cell proliferation and survival in many neoplasms. Results ODAM expression in A375 and C8161 cells strongly inhibited cell growth and motility in vitro, increased cell adhesion to extracellular matrix, and yielded significant cytoskeletal/morphologic rearrangement. Furthermore, AKT activity was downregulated by ODAM expression while an increase was noted in expression of the PTEN (phosphatase and tensin homolog on chromosome 10) tumor suppressor gene, an antagonist of AKT activation. Increased PTEN in ODAM-expressing cells was associated with increases in PTEN mRNA levels and de novo protein synthesis. Silencing of PTEN expression yielded recovery of AKT activity in ODAM-expressing melanoma cells. Similar PTEN elevation and inhibition of AKT by ODAM was observed in MDA-MB-231 breast cancer cells while ODAM expression had no effect in PTEN-deficient BT-549 breast cancer cells. Conclusions The apparent anti-neoplastic effects of ODAM in cultured melanoma and breast cancer cells are associated with increased PTEN expression, and suppression of AKT activity. This association should serve to clarify the clinical import of ODAM expression and any role it may serve as an indicator of tumor behavior. PMID:23648148

  13. Hedgehog-GLI signaling drives self-renewal and tumorigenicity of human melanoma-initiating cells.

    PubMed

    Santini, Roberta; Vinci, Maria C; Pandolfi, Silvia; Penachioni, Junia Y; Montagnani, Valentina; Olivito, Biagio; Gattai, Riccardo; Pimpinelli, Nicola; Gerlini, Gianni; Borgognoni, Lorenzo; Stecca, Barbara

    2012-09-01

    The question of whether cancer stem/tumor-initiating cells (CSC/TIC) exist in human melanomas has arisen in the last few years. Here, we have used nonadherent spheres and the aldehyde dehydrogenase (ALDH) enzymatic activity to enrich for CSC/TIC in a collection of human melanomas obtained from a broad spectrum of sites and stages. We find that melanomaspheres display extensive in vitro self-renewal ability and sustain tumor growth in vivo, generating human melanoma xenografts that recapitulate the phenotypic composition of the parental tumor. Melanomaspheres express high levels of Hedgehog (HH) pathway components and of embryonic pluripotent stem cell factors SOX2, NANOG, OCT4, and KLF4. We show that human melanomas contain a subset of cells expressing high ALDH activity (ALDH(high)), which is endowed with higher self-renewal and tumorigenic abilities than the ALDH(low) population. A good correlation between the number of ALDH(high) cells and sphere formation efficiency was observed. Notably, both pharmacological inhibition of HH signaling by the SMOOTHENED (SMO) antagonist cyclopamine and GLI antagonist GANT61 and stable expression of shRNA targeting either SMO or GLI1 result in a significant decrease in melanoma stem cell self-renewal in vitro and a reduction in the number of ALDH(high) melanoma stem cells. Finally, we show that interference with the HH-GLI pathway through lentiviral-mediated silencing of SMO and GLI1 drastically diminishes tumor initiation of ALDH(high) melanoma stem cells. In conclusion, our data indicate an essential role of the HH-GLI1 signaling in controlling self-renewal and tumor initiation of melanoma CSC/TIC. Targeting HH-GLI1 is thus predicted to reduce the melanoma stem cell compartment. Copyright © 2012 AlphaMed Press.

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

    PubMed

    Serini, Simona; Zinzi, Antonio; Ottes Vasconcelos, Renata; Fasano, Elena; Riillo, Maria Greca; Celleno, Leonardo; Trombino, Sonia; Cassano, Roberta; Calviello, Gabriella

    2016-11-01

    We previously found that docosahexaenoic acid (DHA), a dietary polyunsaturated fatty acid present at high level in fatty fish, inhibited cell growth and induced differentiation of melanoma cells in vitro by increasing nuclear β-catenin content. An anti-neoplastic role of nuclear β-catenin was suggested in melanoma, and related to the presence in the melanocyte lineage of the microphtalmia transcription factor (MITF), which interferes with the transcription of β-catenin/TCF/LEF pro-invasive target genes. In the present work we investigated if DHA could inhibit the invasive potential of melanoma cells, and if this effect could be related to DHA-induced alterations of the Wnt/β-catenin signaling, including changes in MITF expression. WM115 and WM266-4 human melanoma, and B16-F10 murine melanoma cell lines were used. Cell invasion was evaluated by Wound Healing and Matrigel transwell assays. Protein expression was analyzed by Western Blotting and β-catenin phosphorylation by immunoprecipitation. The role of MITF in the anti-invasive effect of DHA was analyzed by siRNA gene silencing. We found that DHA inhibited anchorage-independent cell growth, reduced their migration/invasion in vitro and down-regulated several Matrix Metalloproteinases (MMP: MMP-2, MT1-MMP and MMP-13), known to be involved in melanoma invasion. We related these effects to the β-catenin increased nuclear expression and PKA-dependent phosphorylation, as well as to the increased expression of MITF. The data obtained further support the potential role of dietary DHA as suppressor of melanoma progression to invasive malignancy through its ability to enhance MITF expression and PKA-dependent nuclear β-catenin phosphorylation. Copyright © 2016. Published by Elsevier Ireland Ltd.

  15. Pigment Production Analysis in Human Melanoma Cells.

    PubMed

    Hopkin, Amelia Soto; Paterson, Elyse K; Ruiz, Rolando; Ganesan, Anand K

    2016-05-25

    The human epidermal melanocyte is a highly specialized pigmented cell that serves to protect the epidermis from ultraviolet (UV) damage through the production of melanin, or melanogenesis. Misregulation in melanogenesis leading to either hyper- or hypo-pigmentation is found in human diseases such as malasma and vitiligo. Current therapies for these diseases are largely unsuccessful and the need for new therapies is necessary. In order to identify genes and or compounds that can alter melanogenesis, methods are required that can detect changes in pigment production as well as expression of key melanogenesis transcription factors and enzymes. Here we describe methods to detect changes in melanogenesis in a human melanoma cell line, MNT-1, by (1) analyzing pigment production by measuring the absorbance of melanin present by spectrophotometry, (2) analyzing transcript expression of potent regulators of melanogenesis by qunatitative reverse-transcription (RT)PCR and (3) analyzing protein expression of potent regulators of melanogenesis by Western blot (WB).

  16. Dacarbazine and the Agonistic TRAIL Receptor-2 Antibody Lexatumumab Induce Synergistic Anticancer Effects in Melanoma

    PubMed Central

    Engesæter, Birgit; Engebraaten, Olav; Flørenes, Vivi Ann; Mælandsmo, Gunhild Mari

    2012-01-01

    Mapatumumab and lexatumumab (targeting death receptor 4 (DR4) and 5 (DR5), respectively) are agonistic TRAIL receptor antibodies that induce apoptosis in a wide range of cancer cells. The potency of mapatumumab and lexatumumab was assessed in mono therapy protocols, and the ability to sensitize for dacarbazine (DTIC) treatment was explored in ten different melanoma cell lines. Our data indicated that melanoma cell lines tend to be resistant to mapatumumab, most likely due to low expression of DR4, while a dose dependent response to lexatumumab was observed. Combining DTIC and lexatumumab induced an additive or synergistic effect on cell death in the various melanoma cell lines. The synergistic effect observed in the FEMX-1 cell line was related to enhanced cleavage of Bid in parallel with elevated expression of the pro-apoptotic proteins Bim, Bax and Bak. Furthermore, the anti-apoptotic proteins Bcl-XL, cIAP-1, XIAP and livin were down regulated. Cleavage of Bid and down regulation of cIAP-2 and livin were observed in vivo. Altogether, these data suggest a change in the balance between pro- and anti-apoptotic proteins favoring induction of apoptosis. In the more therapy resistant cell line, HHMS, no changes in the pro- and anti-apoptotic proteins were observed. FEMX-1 xenografts treated with DTIC and lexatumumab showed reduced growth and increased level of apoptosis compared to the control groups, providing arguments for further evaluation of this combination in melanoma patients. PMID:23029050

  17. Dacarbazine and the agonistic TRAIL receptor-2 antibody lexatumumab induce synergistic anticancer effects in melanoma.

    PubMed

    Engesæter, Birgit; Engebraaten, Olav; Flørenes, Vivi Ann; Mælandsmo, Gunhild Mari

    2012-01-01

    Mapatumumab and lexatumumab (targeting death receptor 4 (DR4) and 5 (DR5), respectively) are agonistic TRAIL receptor antibodies that induce apoptosis in a wide range of cancer cells. The potency of mapatumumab and lexatumumab was assessed in mono therapy protocols, and the ability to sensitize for dacarbazine (DTIC) treatment was explored in ten different melanoma cell lines. Our data indicated that melanoma cell lines tend to be resistant to mapatumumab, most likely due to low expression of DR4, while a dose dependent response to lexatumumab was observed. Combining DTIC and lexatumumab induced an additive or synergistic effect on cell death in the various melanoma cell lines. The synergistic effect observed in the FEMX-1 cell line was related to enhanced cleavage of Bid in parallel with elevated expression of the pro-apoptotic proteins Bim, Bax and Bak. Furthermore, the anti-apoptotic proteins Bcl-XL, cIAP-1, XIAP and livin were down regulated. Cleavage of Bid and down regulation of cIAP-2 and livin were observed in vivo. Altogether, these data suggest a change in the balance between pro- and anti-apoptotic proteins favoring induction of apoptosis. In the more therapy resistant cell line, HHMS, no changes in the pro- and anti-apoptotic proteins were observed. FEMX-1 xenografts treated with DTIC and lexatumumab showed reduced growth and increased level of apoptosis compared to the control groups, providing arguments for further evaluation of this combination in melanoma patients.

  18. Loss of alleles from the distal short arm of chromosome 1 occurs late in melanoma tumor progression

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

    Dracopoli, N.C.; Harnett, P.; Bale, S.J.

    The gene for familial malignant melanoma and its precursor lesion, the dysplastic nevus, has been assigned to a region of the distal short arm of chromosome 1, which is frequently involved in karyotypic abnormalities in melanoma cells. The authors have examined loci on chromosome 1p for loss-of-constitutional heterozygosity in 35 melanomas and 21 melanoma cell lines to analyze the role of these abnormalities in melanocyte transformation. Loss-of-heterozygosity at loci on chromosome 1p was identified in 15/35 (43%) melanomas and 11/21 (52%) melanoma cell lines. Analysis of multiple metastases derived from the same patient and of melanoma and lymphoblastoid samples frommore » a family with hereditary melanoma showed that the loss-of-heterozygosity at loci on distal 1p is a late event in tumor progression, rather than the second mutation that would occur if melanoma were due to a cellular recessive mechanism. Comparisons with neuroblastoma and multiple endocrine neoplasia (MEN2) suggest that the frequent 1p loss-of-heterozygosity in these malignancies is a common late event of neuroectodermal tumor progression.« less

  19. Mitogen-activated Protein Kinase (MAPK) Hyperactivation and Enhanced NRAS Expression Drive Acquired Vemurafenib Resistance in V600E BRAF Melanoma Cells*

    PubMed Central

    Lidsky, Michael; Antoun, Gamil; Speicher, Paul; Adams, Bartley; Turley, Ryan; Augustine, Christi; Tyler, Douglas; Ali-Osman, Francis

    2014-01-01

    Although targeting the V600E activating mutation in the BRAF gene, the most common genetic abnormality in melanoma, has shown clinical efficacy in melanoma patients, response is, invariably, short lived. To better understand mechanisms underlying this acquisition of resistance to BRAF-targeted therapy in previously responsive melanomas, we induced vemurafenib resistance in two V600E BRAF+ve melanoma cell lines, A375 and DM443, by serial in vitro vemurafenib exposure. The resulting approximately 10-fold more vemurafenib-resistant cell lines, A375rVem and D443rVem, had higher growth rates and showed differential collateral resistance to cisplatin, melphalan, and temozolomide. The acquisition of vemurafenib resistance was associated with significantly increased NRAS levels in A375rVem and D443rVem, increased activation of the prosurvival protein, AKT, and the MAPKs, ERK, JNK, and P38, which correlated with decreased levels of the MAPK inhibitor protein, GSTP1. Despite the increased NRAS, whole exome sequencing showed no NRAS gene mutations. Inhibition of all three MAPKs and siRNA-mediated NRAS suppression both reversed vemurafenib resistance significantly in A375rVem and DM443rVem. Together, the results indicate a mechanism of acquired vemurafenib resistance in V600E BRAF+ve melanoma cells that involves increased activation of all three human MAPKs and the PI3K pathway, as well as increased NRAS expression, which, contrary to previous reports, was not associated with mutations in the NRAS gene. The data highlight the complexity of the acquired vemurafenib resistance phenotype and the challenge of optimizing BRAF-targeted therapy in this disease. They also suggest that targeting the MAPKs and/or NRAS may provide a strategy to mitigate such resistance in V600E BRAF+ve melanoma. PMID:25063807

  20. Mitogen-activated protein kinase (MAPK) hyperactivation and enhanced NRAS expression drive acquired vemurafenib resistance in V600E BRAF melanoma cells.

    PubMed

    Lidsky, Michael; Antoun, Gamil; Speicher, Paul; Adams, Bartley; Turley, Ryan; Augustine, Christi; Tyler, Douglas; Ali-Osman, Francis

    2014-10-03

    Although targeting the V600E activating mutation in the BRAF gene, the most common genetic abnormality in melanoma, has shown clinical efficacy in melanoma patients, response is, invariably, short lived. To better understand mechanisms underlying this acquisition of resistance to BRAF-targeted therapy in previously responsive melanomas, we induced vemurafenib resistance in two V600E BRAF+ve melanoma cell lines, A375 and DM443, by serial in vitro vemurafenib exposure. The resulting approximately 10-fold more vemurafenib-resistant cell lines, A375rVem and D443rVem, had higher growth rates and showed differential collateral resistance to cisplatin, melphalan, and temozolomide. The acquisition of vemurafenib resistance was associated with significantly increased NRAS levels in A375rVem and D443rVem, increased activation of the prosurvival protein, AKT, and the MAPKs, ERK, JNK, and P38, which correlated with decreased levels of the MAPK inhibitor protein, GSTP1. Despite the increased NRAS, whole exome sequencing showed no NRAS gene mutations. Inhibition of all three MAPKs and siRNA-mediated NRAS suppression both reversed vemurafenib resistance significantly in A375rVem and DM443rVem. Together, the results indicate a mechanism of acquired vemurafenib resistance in V600E BRAF+ve melanoma cells that involves increased activation of all three human MAPKs and the PI3K pathway, as well as increased NRAS expression, which, contrary to previous reports, was not associated with mutations in the NRAS gene. The data highlight the complexity of the acquired vemurafenib resistance phenotype and the challenge of optimizing BRAF-targeted therapy in this disease. They also suggest that targeting the MAPKs and/or NRAS may provide a strategy to mitigate such resistance in V600E BRAF+ve melanoma. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Capture and On-chip analysis of Melanoma Cells Using Tunable Surface Shear forces

    NASA Astrophysics Data System (ADS)

    Tsao, Simon Chang-Hao; Vaidyanathan, Ramanathan; Dey, Shuvashis; Carrascosa, Laura G.; Christophi, Christopher; Cebon, Jonathan; Shiddiky, Muhammad J. A.; Behren, Andreas; Trau, Matt

    2016-01-01

    With new systemic therapies becoming available for metastatic melanoma such as BRAF and PD-1 inhibitors, there is an increasing demand for methods to assist with treatment selection and response monitoring. Quantification and characterisation of circulating melanoma cells (CMCs) has been regarded as an excellent non-invasive candidate but a sensitive and efficient tool to do these is lacking. Herein we demonstrate a microfluidic approach for melanoma cell capture and subsequent on-chip evaluation of BRAF mutation status. Our approach utilizes a recently discovered alternating current electrohydrodynamic (AC-EHD)-induced surface shear forces, referred to as nanoshearing. A key feature of nanoshearing is the ability to agitate fluid to encourage contact with surface-bound antibody for the cell capture whilst removing nonspecific cells from the surface. By adjusting the AC-EHD force to match the binding affinity of antibodies against the melanoma-associated chondroitin sulphate proteoglycan (MCSP), a commonly expressed melanoma antigen, this platform achieved an average recovery of 84.7% from biological samples. Subsequent staining with anti-BRAFV600E specific antibody enabled on-chip evaluation of BRAFV600E mutation status in melanoma cells. We believe that the ability of nanoshearing-based capture to enumerate melanoma cells and subsequent on-chip characterisation has the potential as a rapid screening tool while making treatment decisions.

  2. DEK oncogene is overexpressed during melanoma progression.

    PubMed

    Riveiro-Falkenbach, Erica; Ruano, Yolanda; García-Martín, Rosa M; Lora, David; Cifdaloz, Metehan; Acquadro, Francesco; Ballestín, Claudio; Ortiz-Romero, Pablo L; Soengas, María S; Rodríguez-Peralto, José L

    2017-03-01

    DEK is an oncoprotein involved in a variety of cellular functions, such as DNA repair, replication, and transcriptional control. DEK is preferentially expressed in actively proliferating and malignant cells, including melanoma cell lines in which DEK was previously demonstrated to play a critical role in proliferation and chemoresistance. Still, the impact of this protein in melanoma progression remains unclear. Thus, we performed a comprehensive analysis of DEK expression in different melanocytic tumors. The immunostaining results of 303 tumors demonstrated negligible DEK expression in benign lesions. Conversely, malignant lesions, particularly in metastatic cases, were largely positive for DEK expression, which was partially associated with genomic amplification. Importantly, DEK overexpression was correlated with histological features of aggressiveness in primary tumors and poor prognosis in melanoma patients. In conclusion, our study provides new insight into the involvement of DEK in melanoma progression, as well as proof of concept for its potential application as a marker and therapeutic target of melanoma. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-01-15

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

  5. Therapeutic potential of the metabolic modulator phenformin in targeting the stem cell compartment in melanoma.

    PubMed

    Petrachi, Tiziana; Romagnani, Alessandra; Albini, Adriana; Longo, Caterina; Argenziano, Giuseppe; Grisendi, Giulia; Dominici, Massimo; Ciarrocchi, Alessia; Dallaglio, Katiuscia

    2017-01-24

    Melanoma is the most dangerous and treatment-resistant skin cancer. Tumor resistance and recurrence are due to the persistence in the patient of aggressive cells with stem cell features, the cancer stem cells (CSC). Recent evidences have shown that CSC display a distinct metabolic profile as compared to tumor bulk population: a promising anti-tumor strategy is therefore to target specific metabolic pathways driving CSC behavior. Biguanides (metformin and phenformin) are anti-diabetic drugs able to perturb cellular metabolism and displaying anti-cancer activity. However, their ability to target the CSC compartment in melanoma is not known. Here we show that phenformin, but not metformin, strongly reduces melanoma cell viability, growth and invasion in both 2D and 3D (spheroids) models. While phenformin decreases melanoma CSC markers expression and the levels of the pro-survival factor MITF, MITF overexpression fails to prevent phenformin effects. Phenformin significantly reduces cell viability in melanoma by targeting both CSC (ALDHhigh) and non-CSC cells and by significantly reducing the number of viable cells in ALDHhigh and ALDHlow-derived spheroids. Consistently, phenformin reduces melanoma cell viability and growth independently from SOX2 levels. Our results show that phenformin is able to affect both CSC and non-CSC melanoma cell viability and growth and suggests its potential use as anti-cancer therapy in melanoma.

  6. Therapeutic potential of the metabolic modulator phenformin in targeting the stem cell compartment in melanoma

    PubMed Central

    Albini, Adriana; Longo, Caterina; Argenziano, Giuseppe; Grisendi, Giulia; Dominici, Massimo; Ciarrocchi, Alessia; Dallaglio, Katiuscia

    2017-01-01

    Melanoma is the most dangerous and treatment-resistant skin cancer. Tumor resistance and recurrence are due to the persistence in the patient of aggressive cells with stem cell features, the cancer stem cells (CSC). Recent evidences have shown that CSC display a distinct metabolic profile as compared to tumor bulk population: a promising anti-tumor strategy is therefore to target specific metabolic pathways driving CSC behavior. Biguanides (metformin and phenformin) are anti-diabetic drugs able to perturb cellular metabolism and displaying anti-cancer activity. However, their ability to target the CSC compartment in melanoma is not known. Here we show that phenformin, but not metformin, strongly reduces melanoma cell viability, growth and invasion in both 2D and 3D (spheroids) models. While phenformin decreases melanoma CSC markers expression and the levels of the pro-survival factor MITF, MITF overexpression fails to prevent phenformin effects. Phenformin significantly reduces cell viability in melanoma by targeting both CSC (ALDHhigh) and non-CSC cells and by significantly reducing the number of viable cells in ALDHhigh and ALDHlow-derived spheroids. Consistently, phenformin reduces melanoma cell viability and growth independently from SOX2 levels. Our results show that phenformin is able to affect both CSC and non-CSC melanoma cell viability and growth and suggests its potential use as anti-cancer therapy in melanoma. PMID:28036292

  7. TGF-β-Induced Transcription Sustains Amoeboid Melanoma Migration and Dissemination

    PubMed Central

    Cantelli, Gaia; Orgaz, Jose L.; Rodriguez-Hernandez, Irene; Karagiannis, Panagiotis; Maiques, Oscar; Matias-Guiu, Xavier; Nestle, Frank O.; Marti, Rosa M.; Karagiannis, Sophia N.; Sanz-Moreno, Victoria

    2015-01-01

    Summary Cell migration underlies metastatic dissemination of cancer cells, and fast “amoeboid” migration in the invasive fronts of tumors is controlled by high levels of actomyosin contractility. How amoeboid migration is regulated by extracellular signals and sustained over time by transcriptional changes is not fully understood. Transforming growth factor β (TGF-β) is well known to promote epithelial-to-mesenchymal transition (EMT) and contribute to metastasis, but melanocytes are neural crest derivatives that have undergone EMT during embryonic development. Surprisingly, we find that in melanoma, TGF-β promotes amoeboid features such as cell rounding, membrane blebbing, high levels of contractility, and increased invasion. Using genome-wide transcriptomics, we find that amoeboid melanoma cells are enriched in a TGF-β-driven signature. We observe that downstream of TGF-β, SMAD2 and its adaptor CITED1 control amoeboid behavior by regulating the expression of key genes that activate contractile forces. Moreover, CITED1 is highly upregulated during melanoma progression, and its high expression is associated with poor prognosis. CITED1 is coupled to a contractile-rounded, amoeboid phenotype in a panel of 16 melanoma cell lines, in mouse melanoma xenografts, and in 47 human melanoma patients. Its expression is also enriched in the invasive fronts of lesions. Functionally, we show how the TGF-β-SMAD2-CITED1 axis promotes different steps associated with progression: melanoma detachment from keratinocytes, 2D and 3D migration, attachment to endothelial cells, and in vivo lung metastatic initial colonization and outgrowth. We propose a novel mechanism by which TGF-β-induced transcription sustains actomyosin force in melanoma cells and thereby promotes melanoma progression independently of EMT. PMID:26526369

  8. Metastatic potential of melanoma cells is not affected by electrochemotherapy.

    PubMed

    Todorovic, Vesna; Sersa, Gregor; Mlakar, Vid; Glavac, Damjan; Flisar, Karel; Cemazar, Maja

    2011-06-01

    Electrochemotherapy is a local treatment combining chemotherapy and application of electric pulses to the tumour. Electrochemotherapy with bleomycin and cisplatin has shown its effectiveness in controlling local tumour growth in the treatment of malignant melanoma. However, the effect of electrochemotherapy on the metastatic potential of tumour cells is not known. Prevention of metastasis is an important aspect of successful treatment; however, it is known that metastasis can be induced by different treatment modalities. Therefore, the aim of this study was to evaluate the effect of electrochemotherapy with cisplatin on the metastatic potential of human malignant melanoma cells. Cells treated by electrochemotherapy with cisplatin were tested for their ability to migrate and invade through Matrigel-coated porous membrane. In addition, RNA was isolated from cells after treatment and differentially expressed genes were investigated by microarray analysis to evaluate the effect of electrochemotherapy with cisplatin on gene expression. There were no significant changes observed in cell migration and invasion of melanoma cells after electrochemotherapy. In addition, there were no changes observed in cell adhesion on Matrigel. Gene expression analysis showed that a very low number of genes were differentially expressed after electrochemotherapy with cisplatin. Two genes, LAMB3 and CD63 involved in cell migration, were both downregulated after electrochemotherapy with cisplatin and the expression of metastasis promoting genes was not increased after electrochemotherapy. Our data suggest that electrochemotherapy does not increase the metastatic behaviour of human melanoma cells.

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

    PubMed Central

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

    2017-01-01

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

  10. Aurora kinase B inhibition reduces the proliferation of metastatic melanoma cells and enhances the response to chemotherapy.

    PubMed

    Porcelli, Letizia; Guida, Gabriella; Quatrale, Anna E; Cocco, Tiziana; Sidella, Letizia; Maida, Immacolata; Iacobazzi, Rosa M; Ferretta, Anna; Stolfa, Diana A; Strippoli, Sabino; Guida, Stefania; Tommasi, Stefania; Guida, Michele; Azzariti, Amalia

    2015-01-27

    The poor response to chemotherapy and the brief response to vemurafenib in metastatic melanoma patients, make the identification of new therapeutic approaches an urgent need. Interestingly the increased expression and activity of the Aurora kinase B during melanoma progression suggests it as a promising therapeutic target. The efficacy of the Aurora B kinase inhibitor barasertib-HQPA was evaluated in BRAF mutated cells, sensitive and made resistant to vemurafenib after chronic exposure to the drug, and in BRAF wild type cells. The drug effectiveness has been evaluated as cell growth inhibition, cell cycle progression and cell migration. In addition, cellular effectors of drug resistance and response were investigated. The characterization of the effectors responsible for the resistance to vemurafenib evidenced the increased expression of MITF or the activation of Erk1/2 and p-38 kinases in the newly established cell lines with a phenotype resistant to vemurafenib. The sensitivity of cells to barasertib-HQPA was irrespective of BRAF mutational status. Barasertib-HQPA induced the mitotic catastrophe, ultimately causing apoptosis and necrosis of cells, inhibited cell migration and strongly affected the glycolytic metabolism of cells inducing the release of lactate. In association i) with vemurafenib the gain in effectiveness was found only in BRAF(V600K) cells while ii) with nab-paclitaxel, the combination was more effective than each drug alone in all cells. These findings suggest barasertib as a new therapeutic agent and as enhancer of chemotherapy in metastatic melanoma treatment.

  11. Microsomal PGE2 synthase-1 regulates melanoma cell survival and associates with melanoma disease progression

    PubMed Central

    Kim, Sun-Hee; Hashimoto, Yuuri; Cho, Sung-Nam; Roszik, Jason; Milton, Denái R.; Dal, Fulya; Kim, Sangwon F.; Menter, David G.; Yang, Peiying; Ekmekcioglu, Suhendan; Grimm, Elizabeth A.

    2016-01-01

    Summary COX-2 and its product PGE2 enhance carcinogenesis and tumor progression, which has been previously reported in melanoma. As most COX inhibitors cause much toxicity, the downstream microsomal PGE2 synthase-1 (mPGES1) is a consideration for targeting. Human melanoma TMAs were employed for testing mPGES1 protein staining intensity and percentage levels and both increased with clinical stage; employing a different Stage III TMA, mPGES1 intensity (not percentage) associated with reduced patient survival. Our results further show that iNOS was also highly expressed in melanoma tissues with high mPGES1 levels, and iNOS-mediated NO promoted mPGES1 expression and PGE2 production. An mPGES1specific inhibitor (CAY10526) as well as siRNA attenuated cell survival and increased apoptosis. CAY10526 significantly suppressed tumor growth and increased apoptosis in melanoma xenografts. Our findings support the value of a prognostic and predictive role for mPGES1, and suggest targeting this molecule in the PGE2 pathway as another avenue toward improving melanoma therapy. PMID:26801201

  12. CD200 is induced by ERK and is a potential therapeutic target in melanoma

    PubMed Central

    Petermann, Kimberly B.; Rozenberg, Gabriela I.; Zedek, Daniel; Groben, Pamela; McKinnon, Karen; Buehler, Christin; Kim, William Y.; Shields, Janiel M.; Penland, Shannon; Bear, James E.; Thomas, Nancy E.; Serody, Jonathan S.; Sharpless, Norman E.

    2007-01-01

    Immune-mediated antitumor responses occur in patients with metastatic melanoma (MM), and therapies designed to augment such responses are clinically beneficial. Despite the immunogenicity of melanoma, immunomodulatory therapies fail in the majority of patients with MM. An inability of DCs to sufficiently activate effector cells may, in part, underlie this failure of the antitumor response seen in most patients. In this work, we show that mutation of N-RAS or B-RAF, signature genetic lesions present in most MMs, potently induced the expression of cell-surface CD200, a repressor of DC function. Employing 2 independent, genome-wide microarray analyses, we identified CD200 as a highly dynamic, downstream target of RAS/RAF/MEK/ERK activation in melanoma. CD200 protein was similarly overexpressed in human melanoma cell lines and primary tumors. CD200 mRNA expression correlated with progression and was higher in melanoma than in other solid tumors or acute leukemia. Melanoma cell lines expressing endogenous CD200 repressed primary T cell activation by DCs, while knockdown of CD200 by shRNA abrogated this immunosuppressive effect. These data indicate that in addition to its effects on growth, survival, and motility, ERK activation in MM attenuates a host antitumor immune response, implicating CD200 and its interaction with the CD200 receptor as a potential therapeutic target for MM. PMID:18008004

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

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

    Zou, Ying; Fan, Guobiao; Wang, Xuemin, E-mail: wangxuemeidr@yeah.net

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

  14. The plasma membrane Ca2+ pump PMCA4b inhibits the migratory and metastatic activity of BRAF mutant melanoma cells.

    PubMed

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

    2017-06-15

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

  15. Specific c-Jun target genes in malignant melanoma.

    PubMed

    Schummer, Patrick; Kuphal, Silke; Vardimon, Lily; Bosserhoff, Anja K; Kappelmann, Melanie

    2016-05-03

    A fundamental event in the development and progression of malignant melanoma is the de-regulation of cancer-relevant transcription factors. We recently showed that c-Jun is a main regulator of melanoma progression and, thus, is the most important member of the AP-1 transcription factor family in this disease. Surprisingly, no cancer-related specific c-Jun target genes in melanoma were described in the literature, so far. Therefore, we focused on pre-existing ChIP-Seq data (Encyclopedia of DNA Elements) of 3 different non-melanoma cell lines to screen direct c-Jun target genes. Here, a specific c-Jun antibody to immunoprecipitate the associated promoter DNA was used. Consequently, we identified 44 direct c-Jun targets and a detailed analysis of 6 selected genes confirmed their deregulation in malignant melanoma. The identified genes were differentially regulated comparing 4 melanoma cell lines and normal human melanocytes and we confirmed their c-Jun dependency. Direct interaction between c-Jun and the promoter/enhancer regions of the identified genes was confirmed by us via ChIP experiments. Interestingly, we revealed that the direct regulation of target gene expression via c-Jun can be independent of the existence of the classical AP-1 (5´-TGA(C/G)TCA-3´) consensus sequence allowing for the subsequent down- or up-regulation of the expression of these cancer-relevant genes. In summary, the results of this study indicate that c-Jun plays a crucial role in the development and progression of malignant melanoma via direct regulation of cancer-relevant target genes and that inhibition of direct c-Jun targets through inhibition of c-Jun is a potential novel therapeutic option for treatment of malignant melanoma.

  16. Establishment and characterization of a normal melanocyte cell line derived from pig skin.

    PubMed

    Julé, Sophia; Bossé, Philippe; Egidy, Giorgia; Panthier, Jean-Jacques

    2003-08-01

    Several minipig strains develop spontaneous malignant melanoma. As a first step toward the analysis of genes involved in the tumoral progression of melanoma in these animal models, we developed culture conditions for pig melanocytes whereby melanocytes from normal epidermis can be isolated directly onto mitotically inactivated keratinocytes in Eagle's minimal essential medium supplemented with fetal calf serum, tetradecanoyl phorbol acetate (TPA) and cholera toxin. We also derived an immortal line of pigmented melanocytes from the epidermis of a healthy Meishan pig. This cell line, designated PigMel, retains differentiation function in culture, dependence on TPA and cholera toxin and a diploid chromosome number. PigMel melanocytes exhibit morphological and molecular characteristics common to normal mammalian skin melanocytes.

  17. Pigmented basal cell carcinoma mimicking a superficial spreading melanoma.

    PubMed

    Hasbún Acuña, Paula; Cullen Aravena, Roberto; Maturana Donaire, César; Ares Mora, Raúl; Porras Kusmanic, Ninoska

    2016-12-20

    Basal cell carcinoma is the most common form of skin cancer, especially in elderly people. Pigmented basal cell carcinoma is a rare subtype and has been described in the literature as a nodular and hyperpigmented lesion; rarely, it can appear as an extensive pigmented plate, which may be clinically indistinguishable from superficial spreading melanoma and Bowen disease. Dermatoscopy has a high sensitivity in the diagnosis of basal cell carcinoma. When Menzies criteria are used; however, the final diagnosis is made by histopathology. The objective of the present report is to analyze the case of a patient with pigmented basal cell carcinoma simulating a superficial spreading melanoma.

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

    PubMed

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

    2015-11-27

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

  19. Eradication of melanomas by targeted elimination of a minor subset of tumor cells

    PubMed Central

    Schmidt, Patrick; Kopecky, Caroline; Hombach, Andreas; Zigrino, Paola; Mauch, Cornelia; Abken, Hinrich

    2011-01-01

    Proceeding on the assumption that all cancer cells have equal malignant capacities, current regimens in cancer therapy attempt to eradicate all malignant cells of a tumor lesion. Using in vivo targeting of tumor cell subsets, we demonstrate that selective elimination of a definite, minor tumor cell subpopulation is particularly effective in eradicating established melanoma lesions irrespective of the bulk of cancer cells. Tumor cell subsets were specifically eliminated in a tumor lesion by adoptive transfer of engineered cytotoxic T cells redirected in an antigen-restricted manner via a chimeric antigen receptor. Targeted elimination of less than 2% of the tumor cells that coexpress high molecular weight melanoma-associated antigen (HMW-MAA) (melanoma-associated chondroitin sulfate proteoglycan, MCSP) and CD20 lastingly eradicated melanoma lesions, whereas targeting of any random 10% tumor cell subset was not effective. Our data challenge the biological therapy and current drug development paradigms in the treatment of cancer. PMID:21282657

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

    PubMed Central

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

    2014-01-01

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

  1. The 14-3-3σ gene promoter is methylated in both human melanocytes and melanoma

    PubMed Central

    2009-01-01

    Background Recent evidence demonstrates that 14-3-3σ acts as a tumor suppressor gene inactivated by methylation of its 5' CpG islands in epithelial tumor cells, while remaining un-methylated in normal human epithelia. The methylation analysis of 14-3-3σ has been largely overlooked in melanoma. Methods The methylation status of 14-3-3σ CpG island in melanocytes and melanoma cells was analyzed by methylation-specific sequencing (MSS) and quantitative methylation-specific PCR (Q-MSP). 14-3-3σ mRNA and protein expression in cell lines was detected by real-time RT-PCR and western blot. Melanoma cells were also treated by 5-aza-2'-deoxycytidine (DAC), a demethylating agent, and/or histone deacetylase inhibitor, Trichostatin A (TSA), to evaluate their effects on 14-3-3σ gene expression. Results 14-3-3σ is hypermethylated in both human melanocytes and most melanoma cells in a lineage-specific manner, resulting in the silencing of 14-3-3σ gene expression and the active induction of 14-3-3σ mRNA and protein expression following treatment with DAC. We also observed a synergistic effect upon gene expression when DAC was combined with TSA. The promoter methylation status of 14-3-3σ was analyzed utilizing Q-MSP in 20 melanoma tissue samples and 10 cell lines derived from these samples, showing that the majority of melanoma samples maintain their hypermethylation status of the 14-3-3σ gene. Conclusion 14-3-3σ is hypermethylated in human melanoma in a cell-linage specific manner. Spontaneous demethylation and re-expression of 14-3-3σ is a rare event in melanoma, indicating 14-3-3σ might have a tentative role in the pathogenesis of melanoma. PMID:19473536

  2. MC1R and cAMP signaling inhibit cdc25B activity and delay cell cycle progression in melanoma cells

    PubMed Central

    Lyons, Jesse; Bastian, Boris C.; McCormick, Frank

    2013-01-01

    The melanocortin 1 receptor (MC1R) mediates the tanning response through induction of cAMP and downstream pigmentary enzymes. Diminished function alleles of MC1R are associated with decreased tanning and increased melanoma risk, which has been attributed to increased rates of mutation. We have found that MC1R or cAMP signaling also directly decreases proliferation in melanoma cell lines. MC1R overexpression, treatment with the MC1R ligand, or treatment with small-molecule activators of cAMP signaling causes delayed progression from G2 into mitosis. This delay is caused by phosphorylation and inhibition of cdc25B, a cyclin dependent kinase 1-activating phosphatase, and is rescued by expression of a cdc25B mutant that cannot be phosphorylated at the serine 323 residue. These results show that MC1R and cAMP signaling can directly inhibit melanoma growth through regulation of the G2/M checkpoint. PMID:23908401

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

    PubMed

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

    2015-01-01

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

  4. Cell cycle-tailored targeting of metastatic melanoma: Challenges and opportunities.

    PubMed

    Haass, Nikolas K; Gabrielli, Brian

    2017-07-01

    The advent of targeted therapies of metastatic melanoma, such as MAPK pathway inhibitors and immune checkpoint antagonists, has turned dermato-oncology from the "bad guy" to the "poster child" in oncology. Current targeted therapies are effective, although here is a clear need to develop combination therapies to delay the onset of resistance. Many antimelanoma drugs impact on the cell cycle but are also dependent on certain cell cycle phases resulting in cell cycle phase-specific drug insensitivity. Here, we raise the question: Have combination trials been abandoned prematurely as ineffective possibly only because drug scheduling was not optimized? Firstly, if both drugs of a combination hit targets in the same melanoma cell, cell cycle-mediated drug insensitivity should be taken into account when planning combination therapies, timing of dosing schedules and choice of drug therapies in solid tumors. Secondly, if the combination is designed to target different tumor cell subpopulations of a heterogeneous tumor, one drug effective in a particular subpopulation should not negatively impact on the other drug targeting another subpopulation. In addition to the role of cell cycle stage and progression on standard chemotherapeutics and targeted drugs, we discuss the utilization of cell cycle checkpoint control defects to enhance chemotherapeutic responses or as targets themselves. We propose that cell cycle-tailored targeting of metastatic melanoma could further improve therapy outcomes and that our real-time cell cycle imaging 3D melanoma spheroid model could be utilized as a tool to measure and design drug scheduling approaches. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  5. Dual MAPK inhibition is an effective therapeutic strategy for a subset of class II BRAF mutant melanoma.

    PubMed

    Dankner, Matthew; Lajoie, Mathieu; Moldoveanu, Dan; Nguyen, Tan-Trieu; Savage, Paul; Rajkumar, Shivshankari; Huang, Xiu; Lvova, Maria; Protopopov, Alexei; Vuzman, Dana; Hogg, David; Park, Morag; Guiot, Marie-Christine; Petrecca, Kevin; Mihalcioiu, Catalin; Watson, Ian R; Siegel, Peter M; Rose, April A N

    2018-06-14

    Dual MAPK pathway inhibition (dMAPKi) with BRAF and MEK inhibitors improves survival in BRAF V600E/K mutant melanoma, but the efficacy of dMAPKi in non-V600 BRAF mutant tumors is poorly understood. We sought to characterize the responsiveness of class II (enhanced kinase activity, dimerization dependent) BRAF mutant melanoma to dMAPKi. Tumors from patients with BRAF WT, V600E (class I) and L597S (class II) metastatic melanoma were used to generate patient-derived-xenografts (PDX). We assembled a panel of melanoma cell lines with class IIa (activation segment) or IIb (p-loop) mutations and compared these to wild-type or V600E/K BRAF mutant cells. Cell lines and PDXs were treated with BRAFi (vemurafenib, dabrafenib, encorafenib, LY3009120), MEKi (cobimetinib, trametinib, binimetinib) or the combination. We identified two patients with BRAF L597S metastatic melanoma who were treated with dMAPKi. BRAFi impaired MAPK signalling and cell growth in class I and II BRAF mutant cells. dMAPKi was more effective than either single MAPKi at inhibiting cell growth in all class II BRAF mutant cells tested. dMAPKi caused tumor regression in two melanoma PDXs with class II BRAF mutations, and prolonged survival of mice with class II BRAF mutant melanoma brain metastases. Two patients with BRAF L597S mutant melanoma clinically responded to dMAPKi. Class II BRAF mutant melanoma are growth inhibited by dMAPKi. Responses to dMAPKi have been observed in two patients with class II BRAF mutant melanoma. This data provides rationale for clinical investigation of dMAPKi in patients with class II BRAF mutant metastatic melanoma. Copyright ©2018, American Association for Cancer Research.

  6. ABCB5 promotes melanoma metastasis through enhancing NF-κB p65 protein stability.

    PubMed

    Wang, Shenghao; Tang, Li; Lin, Junyu; Shen, Zhongliang; Yao, Yikun; Wang, Wei; Tao, Shuai; Gu, Chenjian; Ma, Jie; Xie, Youhua; Liu, Yanfeng

    2017-10-07

    Melanoma is the most aggressive type of skin cancer. Melanoma has an extremely poor prognosis because of its high potential for vascular invasion, metastasis and recurrence. The mechanism of melanoma metastasis is not well understood. ATP-binding cassette sub-family B member 5 (ABCB5) plays a key role in melanoma growth. However, it is uncertain what function ABCB5 may exert in melanoma metastasis. In this report, we for the first time demonstrate ABCB5 as a crucial factor that promotes melanoma metastasis. ABCB5 positive (ABCB5 + ) malignant melanoma initiating cells (MMICs) display a higher metastatic potential compared with ABCB5 negative (ABCB5 - ) melanoma subpopulation. Knockdown of ABCB5 expression reduces melanoma cell migration and invasion in vitro and melanoma pulmonary metastasis in tumor xenograft mice. ABCB5 and NF-κB p65 expression levels are positively correlated in both melanoma tissues and cell lines. Consequently, ABCB5 activates the NF-κB pathway by inhibiting p65 ubiquitination to enhance p65 protein stability. Our finding highlights ABCB5 as a novel pro-metastasis factor and provides a potential therapeutic target for melanoma. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. CXCR1 as a novel target for directing reactive T cells toward melanoma: implications for adoptive cell transfer immunotherapy.

    PubMed

    Sapoznik, Sivan; Ortenberg, Rona; Galore-Haskel, Gilli; Kozlovski, Stav; Levy, Daphna; Avivi, Camila; Barshack, Iris; Cohen, Cyrille J; Besser, Michal J; Schachter, Jacob; Markel, Gal

    2012-10-01

    Adoptive cell transfer therapy with reactive T cells is one of the most promising immunotherapeutic modalities for metastatic melanoma patients. Homing of the transferred T cells to all tumor sites in sufficient numbers is of great importance. Here, we seek to exploit endogenous chemotactic signals in order to manipulate and enhance the directional trafficking of transferred T cells toward melanoma. Chemokine profiling of 15 melanoma cultures shows that CXCL1 and CXCL8 are abundantly expressed and secreted from melanoma cultures. However, the complimentary analysis on 40 melanoma patient-derived tumor-infiltrating lymphocytes (TIL) proves that the corresponding chemokine receptors are either not expressed (CXCR2) or expressed at low levels (CXCR1). Using the in vitro transwell system, we demonstrate that TIL cells preferentially migrate toward melanoma and that endogenously expressing CXCR1 TIL cells are significantly enriched among the migrating lymphocytes. The role of the chemokines CXCL1 and CXCL8 is demonstrated by partial abrogation of this enrichment with anti-CXCL1 and anti-CXCL8 neutralizing antibodies. The role of the chemokine receptor CXCR1 is validated by the enhanced migration of CXCR1-engineered TIL cells toward melanoma or recombinant CXCL8. Cytotoxicity and IFNγ secretion activity are unaltered by CXCR1 expression profile. Taken together, these results mark CXCR1 as a candidate for genetic manipulations to enhance trafficking of adoptively transferred T cells. This approach is complimentary and potentially synergistic with other genetic strategies designed to enhance anti-tumor potency.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2011-01-01

    Melanoma, a potentially lethal skin cancer, is widely thought to be immunogenic in nature. While there has been much focus on T cell-mediated immune responses, limited knowledge exists on the role of mature B cells. We describe an approach, including a cell-based ELISA, to evaluate mature IgG antibody responses to melanoma from human peripheral blood B cells. We observed a significant increase in antibody responses from melanoma patients (n = 10) to primary and metastatic melanoma cells compared to healthy volunteers (n = 10) (P<0.0001). Interestingly, we detected a significant reduction in antibody responses to melanoma with advancing disease stage in our patient cohort (n = 21) (P<0.0001). Overall, 28% of melanoma patient-derived B cell cultures (n = 1,800) compared to 2% of cultures from healthy controls (n = 600) produced antibodies that recognized melanoma cells. Lastly, a patient-derived melanoma-specific monoclonal antibody was selected for further study. This antibody effectively killed melanoma cells in vitro via antibody-mediated cellular cytotoxicity. These data demonstrate the presence of a mature systemic B cell response in melanoma patients, which is reduced with disease progression, adding to previous reports of tumor-reactive antibodies in patient sera, and suggesting the merit of future work to elucidate the clinical relevance of activating humoral immune responses to cancer. PMID:21559411

  11. Tamoxifen inhibits tumor cell invasion and metastasis in mouse melanoma through suppression of PKC/MEK/ERK and PKC/PI3K/Akt pathways

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

    Matsuoka, Hiroshi; Department of Pharmacy, Nara Hospital, Kinki University School of Medicine, 1248-1 Ikoma, Nara 630-0293; Tsubaki, Masanobu

    2009-07-15

    In melanoma, several signaling pathways are constitutively activated. Among these, the protein kinase C (PKC) signaling pathways are activated through multiple signal transduction molecules and appear to play major roles in melanoma progression. Recently, it has been reported that tamoxifen, an anti-estrogen reagent, inhibits PKC signaling in estrogen-negative and estrogen-independent cancer cell lines. Thus, we investigated whether tamoxifen inhibited tumor cell invasion and metastasis in mouse melanoma cell line B16BL6. Tamoxifen significantly inhibited lung metastasis, cell migration, and invasion at concentrations that did not show anti-proliferative effects on B16BL6 cells. Tamoxifen also inhibited the mRNA expressions and protein activities ofmore » matrix metalloproteinases (MMPs). Furthermore, tamoxifen suppressed phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt through the inhibition of PKC{alpha} and PKC{delta} phosphorylation. However, other signal transduction factor, such as p38 mitogen-activated protein kinase (p38MAPK) was unaffected. The results indicate that tamoxifen suppresses the PKC/mitogen-activated protein kinase kinase (MEK)/ERK and PKC/phosphatidylinositol-3 kinase (PI3K)/Akt pathways, thereby inhibiting B16BL6 cell migration, invasion, and metastasis. Moreover, tamoxifen markedly inhibited not only developing but also clinically evident metastasis. These findings suggest that tamoxifen has potential clinical applications for the treatment of tumor cell metastasis.« less

  12. Melanoma Cells Can Adopt the Phenotype of Stromal Fibroblasts and Macrophages by Spontaneous Cell Fusion in Vitro

    PubMed Central

    Kemény, Lajos V.; Kurgyis, Zsuzsanna; Buknicz, Tünde; Groma, Gergely; Jakab, Ádám; Zänker, Kurt; Dittmar, Thomas; Kemény, Lajos; Németh, István B.

    2016-01-01

    After the removal of primary cutaneous melanoma some patients develop local recurrences, even after having histologically tumor-free re-excision. A potential explanation behind this phenomenon is that tumor cells switch their phenotype, making their recognition via standard histopathological assessments extremely difficult. Tumor-stromal cell fusion has been proposed as a potential mechanism for tumor cells to acquire mesenchymal traits; therefore, we hypothesized that melanoma cells could acquire fibroblast- and macrophage-like phenotypes via cell fusion. We show that melanoma cells spontaneously fuse with human dermal fibroblasts and human peripheral blood monocytes in vitro. The hybrid cells’ nuclei contain chromosomes from both parental cells and are indistinguishable from the parental fibroblasts or macrophages based on their morphology and immunophenotype, as they could lose the melanoma specific MART1 marker, but express the fibroblast marker smooth muscle actin or the macrophage marker CD68. Our results suggest that, by spontaneous cell fusion in vitro, tumor cells can adopt the morphology and immunophenotype of stromal cells while still carrying oncogenic, tumor-derived genetic information. Therefore, melanoma–stromal cell fusion might play a role in missing tumor cells by routine histopathological assessments. PMID:27271591

  13. Immunohistochemical detection of XIAP in melanoma.

    PubMed

    Emanuel, Patrick O M; Phelps, Robert G; Mudgil, Adarsh; Shafir, Michail; Burstein, David E

    2008-03-01

    The X-linked inhibitor of apoptosis protein (XIAP) is the most potent of the inhibitor of apoptosis family of eight proteins. High levels of XIAP have been found in melanoma cell lines and are believed to play a role in therapeutic resistance in a number of malignancies. XIAP expression has not been investigated in clinically obtained melanoma tissue samples, nor have studies attempted to correlate XIAP expression with prognostic variables or clinical aggressiveness of melanomas. Sixty-seven patients with primary cutaneous malignant melanoma for whom clinical follow up was available were identified from the records of the Mount Sinai Hospital, comprising 37 thin melanomas (Breslow thickness < 1.0 mm) and 30 thick melanomas (Breslow thickness > 1.0 mm). Archival paraffin sections from primary lesions and corresponding metastases were stained with monoclonal anti-XIAP antibody using routine immunohistochemical methods. Six benign intradermal nevi and four in situ melanomas were XIAP negative. 9 of 37 thin melanomas (24%) were XIAP positive. In contrast, 21 of 30 (73%) thick melanomas were XIAP positive, including 3 of 4 ulcerated melanomas that were strongly positive. Over a follow-up period ranging from 6 months to 6 years, 23 melanomas metastasized (22 thick, 1 thin). In total, XIAP was immunohistochemically detected in 17 of 23 metastases (74%). Metastasis occurred in 1 of 9 XIAP-positive thin melanomas; 0 of 28 XIAP-negative thin melanomas; 17 of 22 XIAP-positive thick melanomas, and 5 of 8 XIAP-negative thick melanomas (63%). XIAP is immunohistochemically detectable nearly three times more frequently in thick compared with thin melanomas. These results suggest that XIAP elevation may be correlated with increasing melanoma thickness and tumor progression.

  14. Vaccination with Irradiated Autologous Melanoma Cells Engineered to Secrete Human Granulocyte--Macrophage Colony-Stimulating Factor Generates Potent Antitumor Immunity in Patients with Metastatic Melanoma

    NASA Astrophysics Data System (ADS)

    Soiffer, Robert; Lynch, Thomas; Mihm, Martin; Jung, Ken; Rhuda, Catherine; Schmollinger, Jan C.; Hodi, F. Stephen; Liebster, Laura; Lam, Prudence; Mentzer, Steven; Singer, Samuel; Tanabe, Kenneth K.; Benedict Cosimi, A.; Duda, Rosemary; Sober, Arthur; Bhan, Atul; Daley, John; Neuberg, Donna; Parry, Gordon; Rokovich, Joseph; Richards, Laurie; Drayer, Jan; Berns, Anton; Clift, Shirley; Cohen, Lawrence K.; Mulligan, Richard C.; Dranoff, Glenn

    1998-10-01

    We conducted a Phase I clinical trial investigating the biologic activity of vaccination with irradiated autologous melanoma cells engineered to secrete human granulocyte--macrophage colony-stimulating factor in patients with metastatic melanoma. Immunization sites were intensely infiltrated with T lymphocytes, dendritic cells, macrophages, and eosinophils in all 21 evaluable patients. Although metastatic lesions resected before vaccination were minimally infiltrated with cells of the immune system in all patients, metastatic lesions resected after vaccination were densely infiltrated with T lymphocytes and plasma cells and showed extensive tumor destruction (at least 80%), fibrosis, and edema in 11 of 16 patients examined. Antimelanoma cytotoxic T cell and antibody responses were associated with tumor destruction. These results demonstrate that vaccination with irradiated autologous melanoma cells engineered to secrete granulocyte--macrophage colony-stimulating factor stimulates potent antitumor immunity in humans with metastatic melanoma.

  15. O(6)-methylguanine DNA-methyltransferase (MGMT) overexpression in melanoma cells induces resistance to nitrosoureas and temozolomide but sensitizes to mitomycin C.

    PubMed

    Passagne, Isabelle; Evrard, Alexandre; Depeille, Philippe; Cuq, Pierre; Cupissol, Didier; Vian, Laurence

    2006-03-01

    Alkylating agents play an important role in the chemotherapy of malignant melanomas. The activity of alkylating agents depends on their capacity to form alkyl adducts with DNA, in some cases causing cross-linking of DNA strands. However, the use of these agents is limited by cellular resistance induced by the DNA repair enzyme O(6)-methylguanine DNA-methyltransferase (MGMT) which removes alkyl groups from alkylated DNA strands. To determine to what extent the expression of MGMT in melanoma cells induces resistance to alkylating agents, the human cell line CAL77 Mer- (i.e., MGMT deficient) were transfected with pcMGMT vector containing human MGMT cDNA. Several clones expressing MGMT at a high level were selected to determine their sensitivity to chemotherapeutic drugs. Melanoma-transfected cells were found to be significantly less sensitive to nitrosoureas (carmustine, fotemustine, streptozotocin) and temozolomide with an increase of IC(50) values between 3 and 14 when compared to parent cells. No difference in cell survival rates between MGMT-proficient and -deficient cells was observed for melphalan, chlorambucil, busulphan, thiotepa and cisplatin which preferentially induce N(7) guanine lesions. Surprisingly, MGMT overexpression increased the sensitivity of CAL77 cells to mitomycin C by approximately 10-fold. Treatment of clonal cell lines with buthionine-[S,R]-sulfoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase which depletes cellular glutathione, completely reversed this unexpected increase in sensitivity to mitomycin C. This observation suggests that glutathione is involved in the sensitivity of MGMT-transfected cells to mitomycin C and may act synergistically with MGMT via an unknown mechanism.

  16. Dihydroxyacetone induces G2/M arrest and apoptotic cell death in A375P melanoma cells.

    PubMed

    Smith, Kelly R; Granberry, Molley; Tan, Marcus C B; Daniel, Casey L; Gassman, Natalie R

    2018-03-01

    The active ingredient in sunless tanning products (STPs) is a simple sugar, dihydroxyacetone (DHA). Several studies have demonstrated that DHA is absorbed within the viable layers of skin and not fully contained within the stratum corneum. Additionally, spray tanning and other aerosolized application methods have increased the risk of internal exposure through mucous membranes and inhalation. Beyond its presence in STPs, DHA also occurs as an endogenous by-product of fructose metabolism, and an excess of DHA in cells can induce advanced glycation end (AGE) products and oxidative stress. Therefore, exogenous and endogenous exposures to DHA may be harmful to cells, and it has already been demonstrated that exogenous exposure to DHA is cytotoxic in immortalized keratinocytes. Still, little is known about the exogenous DHA exposure effects on other skin components. In this study, we explore the effects of exogenous DHA exposure in a human melanoma cell line, A375P. Melanoma cells were sensitive to DHA and displayed a transient burst of reactive oxygen species within an hour of exposure. Cell cycle arrest at G2/M was observed within 24 h of exposure, and apoptosis, monitored by the cleavage of PARP-1 and Caspase-3, was detected within 72 h of exposure to DHA. Together, these demonstrate that exogenous exposure to DHA has cytotoxic effects in our selected cell model and indicates the need to further investigate the exogenous exposure effects of DHA in other relevant exposure models. © 2017 Wiley Periodicals, Inc.

  17. Epigenetic regulation of the transcription factor Foxa2 directs differential elafin expression in melanocytes and melanoma cells

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

    Yu, Kyung Sook; Jo, Ji Yoon; Kim, Su Jin

    2011-04-29

    Highlights: {yields} Elafin expression is epigenetically silenced in human melanoma cells. {yields} Foxa2 expression in melanoma cells is silenced by promoter hypermethylation. {yields} Foxa2 directs activation of the elafin promoter in vivo. {yields} Foxa2 expression induces apoptosis of melanoma cells via elafin re-expression. -- Abstract: Elafin, a serine protease inhibitor, induces the intrinsic apoptotic pathway in human melanoma cells, where its expression is transcriptionally silenced. However, it remains unknown how the elafin gene is repressed in melanoma cells. We here demonstrate that elafin expression is modulated via epigenetically regulated expression of the transcription factor Foxa2. Treatment of melanoma cells withmore » a DNA methyltransferase inhibitor induced elafin expression, which was specifically responsible for reduced proliferation and increased apoptosis. Suppression of Foxa2 transcription, mediated by DNA hypermethylation in its promoter region, was released in melanoma cells upon treatment with the demethylating agent. Luciferase reporter assays indicated that the Foxa2 binding site in the elafin promoter was critical for the activation of the promoter. Chromatin immunoprecipitation assays further showed that Foxa2 bound to the elafin promoter in vivo. Analyses of melanoma cells with varied levels of Foxa2 revealed a correlated expression between Foxa2 and elafin and the ability of Foxa2 to induce apoptosis. Our results collectively suggest that, in melanoma cells, Foxa2 expression is silenced and therefore elafin is maintained unexpressed to facilitate cell proliferation in the disease melanoma.« less

  18. CD133+ cell content correlates with tumour growth in melanomas from skin with chronic sun-induced damage.

    PubMed

    González-Herrero, I; Romero-Camarero, I; Cañueto, J; Cardeñoso-Álvarez, E; Fernández-López, E; Pérez-Losada, J; Sánchez-García, I; Román-Curto, C

    2013-10-01

    Melanoma is responsible for almost 80% of the deaths attributed to skin cancer. Stem cells, defined by CD133 expression, have been implicated in melanoma tumour growth, but their specific role is still uncertain. We hypothesized that the phenotypic heterogeneity of human cutaneous melanomas is related to their content of CD133+ cells. We compared the percentages of CD133+ cells in 29 tumours from four classic types of melanoma: lentigo maligna melanoma (LMM), superficial spreading melanoma, nodular melanoma and acral lentiginous melanoma (ALM). Also, we compared the percentages of CD133+ cells in melanomas with different degrees of exposure to ultraviolet radiation: 16 melanomas from skin with chronic sun-induced damage and 13 melanomas from skin without such damage. We found a statistically significant increase of CD133+ cells in three different contexts: in melanomas arising on skin with signs of chronic sun-induced damage vs. nonexposed skin, in melanomas in situ vs. invasive melanomas, and in LMM vs. ALM. The proportions of CD133+ cells did not differ among samples of normal skin with different degrees of sun exposure. A distinct subpopulation of CD133+CXCR4+ cancer stem cells (CSCs) was identified and shown to be related to the invasive phenotype of the tumours. Here, we provide evidence showing, for the first time, that an increase in the CD133+ cell content is associated both with melanomas arising on skin with signs of chronic sun-induced damage and in melanomas in situ with better prognosis. Moreover, our study further confirms the existence of a subpopulation of CD133+CXCR4+ CSCs in cutaneous melanomas with invasive phenotype and poor prognosis. © 2013 British Association of Dermatologists.

  19. Growth-inhibitory and antiangiogenic activity of the MEK inhibitor PD0325901 in malignant melanoma with or without BRAF mutations.

    PubMed

    Ciuffreda, Ludovica; Del Bufalo, Donatella; Desideri, Marianna; Di Sanza, Cristina; Stoppacciaro, Antonella; Ricciardi, Maria Rosaria; Chiaretti, Sabina; Tavolaro, Simona; Benassi, Barbara; Bellacosa, Alfonso; Foà, Robin; Tafuri, Agostino; Cognetti, Francesco; Anichini, Andrea; Zupi, Gabriella; Milella, Michele

    2009-08-01

    The Raf/MEK/ERK pathway is an important mediator of tumor cell proliferation and angiogenesis. Here, we investigated the growth-inhibitory and antiangiogenic properties of PD0325901, a novel MEK inhibitor, in human melanoma cells. PD0325901 effects were determined in a panel of melanoma cell lines with different genetic aberrations. PD0325901 markedly inhibited ERK phosphorylation and growth of both BRAF mutant and wild-type melanoma cell lines, with IC(50) in the nanomolar range even in the least responsive models. Growth inhibition was observed both in vitro and in vivo in xenograft models, regardless of BRAF mutation status, and was due to G(1)-phase cell cycle arrest and subsequent induction of apoptosis. Cell cycle (cyclin D1, c-Myc, and p27(KIP1)) and apoptosis (Bcl-2 and survivin) regulators were modulated by PD0325901 at the protein level. Gene expression profiling revealed profound modulation of several genes involved in the negative control of MAPK signaling and melanoma cell differentiation, suggesting alternative, potentially relevant mechanisms of action. Finally, PD0325901 inhibited the production of the proangiogenic factors vascular endothelial growth factor and interleukin 8 at a transcriptional level. In conclusion, PD0325901 exerts potent growth-inhibitory, proapoptotic, and antiangiogenic activity in melanoma lines, regardless of their BRAF mutation status. Deeper understanding of the molecular mechanisms of action of MEK inhibitors will likely translate into more effective treatment strategies for patients experiencing malignant melanoma.

  20. Evaluation of antitumor effects of folate-conjugated methyl-β-cyclodextrin in melanoma.

    PubMed

    Motoyama, Keiichi; Onodera, Risako; Tanaka, Nao; Kameyama, Kazuhisa; Higashi, Taishi; Kariya, Ryusho; Okada, Seiji; Arima, Hidetoshi

    2015-01-01

    Melanoma is a life-threatening disorder and its incidence is increasing gradually. Despite the numerous treatment approaches, conventional systemic chemotherapy has not reduced the mortality rate among melanoma patients, probably due to the induction of toxicity to normal tissues. Recently, we have developed folate-conjugated methyl-β-cyclodextrin (FA-M-β-CyD) and clarified its potential as a new antitumor agent involved in autophagic cell death. However, it remains uncertain whether FA-M-β-CyD exerts anticancer effects against melanomas. Therefore, in this study, we investigated the effects of FA-M-β-CyD on the folate receptor-α (FR-α)-expressing melanoma cell-selective cytotoxic effect. FA-M-β-CyD showed cytotoxic effects in Ihara cells, a human melanoma cell line expressing FR-α. In sharp contrast to methyl-β-cyclodextrin, FA-M-β-CyD entered Ihara cells [FR-α(+)] through FR-α-mediated endocytosis. Additionally, FA-M-β-CyD elicited the formation of autophagosomes in Ihara cells. Notably, FA-M-β-CyD suppressed melanoma growth in BALB/c nude recombinase-activating gene-2 (Rag-2)/Janus kinase 3 (Jak3) double deficient mice bearing Ihara cells. Therefore, these results suggest that FA-M-β-CyD could be utilized as a potent anticancer agent for melanoma chemotherapy by regulating autophagy.

  1. Lansoprazole and carbonic anhydrase IX inhibitors sinergize against human melanoma cells.

    PubMed

    Federici, Cristina; Lugini, Luana; Marino, Maria Lucia; Carta, Fabrizio; Iessi, Elisabetta; Azzarito, Tommaso; Supuran, Claudiu T; Fais, Stefano

    2016-01-01

    Proton Pump Inhibitors (PPIs) reduce tumor acidity and therefore resistance of tumors to drugs. Carbonic Anhydrase IX (CA IX) inhibitors have proven to be effective against tumors, while tumor acidity might impair their full effectiveness. To analyze the effect of PPI/CA IX inhibitors combined treatment against human melanoma cells. The combination of Lansoprazole (LAN) and CA IX inhibitors (FC9-399A and S4) has been investigated in terms of cell proliferation inhibition and cell death in human melanoma cells. The combination of these inhibitors was more effective than the single treatments in both inhibiting cell proliferation and in inducing cell death in human melanoma cells. These results represent the first successful attempt in combining two different proton exchanger inhibitors. This is the first evidence on the effectiveness of a new approach against tumors based on the combination of PPI and CA IX inhibitors, thus providing an alternative strategy against tumors.

  2. Mutated PPP1R3B is recognized by T cells used to treat a melanoma patient who experienced a durable complete tumor regression

    PubMed Central

    Lu, Yong-Chen; Yao, Xin; Li, Yong F.; El-Gamil, Mona; Dudley, Mark E.; Yang, James C.; Almeida, Jorge R.; Douek, Daniel C.; Samuels, Yardena; Rosenberg, Steven A.; Robbins, Paul F.

    2013-01-01

    Adoptive cell therapy with tumor infiltrating lymphocytes (TILs) represents an effective treatment for patients with metastatic melanoma. However, most of the antigen targets recognized by effective melanoma reactive TILs remain elusive. In this study, patient 2369 experienced a complete response, including regressions of bulky liver tumor masses ongoing beyond seven years following adoptive TILs transfer. The screening of a cDNA library generated from the autologous melanoma cell line resulted in the isolation of a mutated PPP1R3B (protein phosphatase 1, regulatory (inhibitor) subunit 3B) gene product. The mutated PPP1R3B peptide represents the immunodominant epitope recognized by tumor reactive T cells in TIL 2369. Five years following adoptive transfer, peripheral blood T lymphocytes obtained from patient 2369 recognized the mutated PPP1R3B epitope. These results demonstrate that adoptive T cell therapy targeting a tumor-specific antigen can mediate long-term survival for a patient with metastatic melanoma. This study also provides an impetus to develop personalized immunotherapy targeting tumor-specific, mutated antigens. PMID:23690473

  3. Microsomal PGE2 synthase-1 regulates melanoma cell survival and associates with melanoma disease progression.

    PubMed

    Kim, Sun-Hee; Hashimoto, Yuuri; Cho, Sung-Nam; Roszik, Jason; Milton, Denái R; Dal, Fulya; Kim, Sangwon F; Menter, David G; Yang, Peiying; Ekmekcioglu, Suhendan; Grimm, Elizabeth A

    2016-05-01

    COX-2 and its product PGE2 enhance carcinogenesis and tumor progression, which has been previously reported in melanoma. As most COX inhibitors cause much toxicity, the downstream microsomal PGE2 synthase-1 (mPGES1) is a consideration for targeting. Human melanoma TMAs were employed for testing mPGES1 protein staining intensity and percentage levels, and both increased with clinical stage; employing a different Stage III TMA, mPGES1 intensity (not percentage) associated with reduced patient survival. Our results further show that iNOS was also highly expressed in melanoma tissues with high mPGES1 levels, and iNOS-mediated NO promoted mPGES1 expression and PGE2 production. An mPGES1-specific inhibitor (CAY10526) as well as siRNA attenuated cell survival and increased apoptosis. CAY10526 significantly suppressed tumor growth and increased apoptosis in melanoma xenografts. Our findings support the value of a prognostic and predictive role for mPGES1, and suggest targeting this molecule in the PGE2 pathway as another avenue toward improving melanoma therapy. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  4. Aggressive melanoma cells escape from BMP7-mediated autocrine growth inhibition through coordinated Noggin upregulation

    PubMed Central

    Hsu, Mei-Yu; Rovinsky, Sherry; Lai, Chiou-Yan; Qasem, Shadi; Liu, Xiaoming; How, Joan; Engelhardt, John F.; Murphy, George F.

    2009-01-01

    Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily responsible for mediating a diverse array of cellular functions both during embryogenesis and in adult life. Previously, we reported that upregulation of BMP7 in human melanoma correlates with tumor progression. However, melanoma cells are either inhibited by or become resistant to BMP7 as a function of tumor progression, with normal melanocytes being most susceptible. Herein, real-time quantitative reverse transcriptase-polymerase chain reactions and Western blotting revealed that the expression of BMP antagonist, Noggin, correlates with resistance to BMP7 in advanced melanoma cells. To test the hypothesis that coordinated upregulation of Noggin protects advanced melanoma cells from autocrine inhibition by BMP7, functional expression of Noggin in susceptible melanoma cells was achieved by adenoviral gene transfer. The Noggin-overexpressing cells exhibited a growth advantage in response to subsequent BMP7 transduction in vitro under anchorage-dependent and -independent conditions, in three-dimensional skin reconstructs, as well as in vivo in severe combined immune-deficiency mice. In concordance, Noggin knockdown by lentiviral shRNA confers sensitivity to BMP7-induced growth inhibition in advanced melanoma cells. Our findings suggest that, like TGF-β, BMP7 acts as an autocrine growth inhibitor in melanocytic cells, and that advanced melanoma cells may escape from BMP7-induced inhibition through concomitant aberrant expression of Noggin. PMID:18560367

  5. Identification of glycogen synthase kinase 3α as a therapeutic target in melanoma

    PubMed Central

    Madhunapantula, SubbaRao V.; Sharma, Arati; Gowda, Raghavendra; Robertson, Gavin P.

    2014-01-01

    Summary Deregulated expression or activity of kinases can lead to melanomas, but often the particular kinase isoform causing the effect is not well established, making identification and validation of different isoforms regulating disease development especially important. To accomplish this objective, an siRNA screen was undertaken that which identified glycogen synthase kinase 3α (GSK3α) as an important melanoma growth regulator. Melanocytes and melanoma cell lines representing various stages of melanoma tumor progression expressed both GSK3α and GSK3β, but analysis of tumors in patients with melanoma showed elevated expression of GSK3α in 72% of samples, which was not observed for GSK3β. Furthermore, 80% of tumors in patients with melanoma expressed elevated levels of catalytically active phosphorylated GSK3α (pGSK3αY279), but not phosphorylated GSK3β (pGSK3βY216). siRNA-mediated reduction in GSK3α protein levels reduced melanoma cell survival and proliferation, sensitized cells to apoptosis-inducing agents and decreased xenografted tumor development by up to 56%. Mechanistically, inhibiting GSK3α expression using siRNA or the pharmacological agent AR-A014418 arrested melanoma cells in the G0/G1 phase of the cell cycle and induced apoptotic death to retard tumorigenesis. Therefore, GSK3α is a key therapeutic target in melanoma. PMID:24034838

  6. Adoptive Cell Therapy for Patients with Melanoma

    PubMed Central

    Dudley, Mark E.

    2011-01-01

    Adoptive cell therapy can be an effective treatment for some patients with advanced cancer. This report summarizes clinical trial results from the Surgery Branch, NCI, investigating tumor infiltrating lymphocytes (TIL) and gene engineered peripheral blood T cells for the therapy of patients with melanoma and other solid tumors. PMID:21716716

  7. Selenium nanoparticles fabricated in Undaria pinnatifida polysaccharide solutions induce mitochondria-mediated apoptosis in A375 human melanoma cells.

    PubMed

    Chen, Tianfeng; Wong, Yum-Shing; Zheng, Wenjie; Bai, Yan; Huang, Liang

    2008-11-15

    Selenium nanoparticle (Nano-Se) is a novel Se species with novel biological activities and low toxicity. In the present study, we demonstrated a simple method for synthesis of size-controlled Nano-Se by adding Undaria pinnatifida polysaccharides to the redox system of selenite and ascorbic acid. A panel of four human cancer cell lines was shown to be susceptible to Nano-Se, with IC(50) values ranging from 3.0 to 14.1 microM. Treatment of A375 human melanoma cells with the Nano-Se resulted in dose-dependent cell apoptosis as indicated by DNA fragmentation and phosphatidylserine translocation. Further investigation on intracellular mechanisms found that Nano-Se treatment triggered apoptotic cell death in A375 cells with the involvement of oxidative stress and mitochondrial dysfunction. Our results suggest that Nano-Se may be a candidate for further evaluation as a chemopreventive and chemotherapeutic agent for human cancers, especially melanoma cancer.

  8. Uptake and metabolism of boronophenylalanine in human uveal melanoma cells in culture Relevance to boron neutron capture therapy of cancer cells.

    PubMed

    Belkhou, R; Abbé, J C; Pham, P; Jasner, N; Sahel, J; Dreyfus, H; Moutaouakkil, M; Massarelli, R

    1995-06-01

    The transport of boronophenylalanine (BPA) and its metabolic fate have been studied in a human uveal melanoma cell line isolated from a primary enucleated tumor. The boronated compound was rapidly incorporated into the cells reaching a peak of incorporation in two hours. This was followed by a trough between 10 and 24 hours and by an increase thereafter. The analogy with the amino acids phenylalanine (Phe) and tyrosine (Tyr) was studied in competition experiments incubating cultures of cell line MK-T, isolated in this laboratory, with [(3)H]-Phe and [(125)I]-Tyr, in the presence or absence of various concentrations of BPA, between 0 and 5 min. The presence of BPA severely reduced the uptake of both amino acids. The kinetics of the transport of [(3)H]-Phe and [(3)H]-Tyr in the presence of BPA, measured after 10 sec of incubation, showed that the boronated compound exerted a competitive inhibition on both transport systems. The intracellular metabolism of BPA was followed by measuring boron concentration (measured with Ionization Coupled Mass Spectrometry) in subcellular fractions and after membrane extraction by the detergent Triton X-100. The results showed that BPA remained in the supernatant and was not metabolized into macromolecules. These results and the relative absence of melanine in these cells, as observed by electron microscopy, suggest that BPA may be actively transported into melanoma cells but not metabolized. The results may have a relevance in studies on Boron Neutron Capture Therapy.

  9. Human Papilloma Virus in Melanoma Biopsy Specimens and Its Relation to Melanoma Progression

    PubMed Central

    Dréau, Didier; Culberson, Cathy; Wyatt, Sharon; Holder, Walter D.

    2000-01-01

    Objectives To evaluate melanoma biopsy specimens for human papilloma virus (HPV) and determine the relation between the presence of HPV, in vitro growth, and clinical progression of melanoma in the patients from whom the biopsy specimens were derived. Summary Background Data Ultraviolet radiation from sun exposure appears to be the primary causal agent in the development of cutaneous melanoma. However, other agents, including HPV, as observed in different epithelial carcinomas, may also play a role in melanoma development and progression. Methods Twelve melanoma biopsy specimens obtained from 12 patients with AJCC stage III and IV melanoma were stained with antibodies against gp-100 (HMB-45) and S-100 protein to confirm melanoma diagnosis and with a polyclonal HPV antibody. After mechanical dissociation, the melanoma specimen cells’ ability to grow in vitro was assessed. Patients were evaluated for melanoma progression with physical examination, complete blood count, and liver function tests every 3 months and a chest radiograph every 6 months. Results All biopsy specimens were positive for S-100, and nine (75%) were positive for gp-100. Seven of 12 (58%) were positive for HPV by immunohistochemistry. In vitro, none of the HPV-negative tumor cells grew from the tumor biopsies, whereas five of seven (71%) of the HPV-positive melanoma tumor cells grew very well. All patients with HPV-positive tumor cells had recurrences and died of melanoma progression, whereas four of five (80%) patients with HPV-negative tumor cells remained alive and without melanoma recurrence. Conclusions The presence of HPV was found in 58% of the biopsy specimens obtained from patients with stage III and IV melanoma and correlated with rapid melanoma progression. HPV may serve as a cofactor in the development of melanoma and may modulate a more aggressive phenotype in HPV-containing melanoma cells. PMID:10767787

  10. Growth-Inhibitory and Antiangiogenic Activity of the MEK Inhibitor PD0325901 in Malignant Melanoma with or without BRAF Mutations12

    PubMed Central

    Ciuffreda, Ludovica; Del Bufalo, Donatella; Desideri, Marianna; Di Sanza, Cristina; Stoppacciaro, Antonella; Ricciardi, Maria Rosaria; Chiaretti, Sabina; Tavolaro, Simona; Benassi, Barbara; Bellacosa, Alfonso; Foà, Robin; Tafuri, Agostino; Cognetti, Francesco; Anichini, Andrea; Zupi, Gabriella; Milella, Michele

    2009-01-01

    The Raf/MEK/ERK pathway is an important mediator of tumor cell proliferation and angiogenesis. Here, we investigated the growth-inhibitory and antiangiogenic properties of PD0325901, a novel MEK inhibitor, in human melanoma cells. PD0325901 effects were determined in a panel of melanoma cell lines with different genetic aberrations. PD0325901 markedly inhibited ERK phosphorylation and growth of both BRAF mutant and wild-type melanoma cell lines, with IC50 in the nanomolar range even in the least responsive models. Growth inhibition was observed both in vitro and in vivo in xenograft models, regardless of BRAF mutation status, and was due to G1-phase cell cycle arrest and subsequent induction of apoptosis. Cell cycle (cyclin D1, c-Myc, and p27KIP1) and apoptosis (Bcl-2 and survivin) regulators were modulated by PD0325901 at the protein level. Gene expression profiling revealed profound modulation of several genes involved in the negative control of MAPK signaling and melanoma cell differentiation, suggesting alternative, potentially relevant mechanisms of action. Finally, PD0325901 inhibited the production of the proangiogenic factors vascular endothelial growth factor and interleukin 8 at a transcriptional level. In conclusion, PD0325901 exerts potent growth-inhibitory, proapoptotic, and antiangiogenic activity in melanoma lines, regardless of their BRAF mutation status. Deeper understanding of the molecular mechanisms of action of MEK inhibitors will likely translate into more effective treatment strategies for patients experiencing malignant melanoma. PMID:19649202

  11. Genomic analysis and clinical management of adolescent cutaneous melanoma.

    PubMed

    Rabbie, Roy; Rashid, Mamunur; Arance, Ana M; Sánchez, Marcelo; Tell-Marti, Gemma; Potrony, Miriam; Conill, Carles; van Doorn, Remco; Dentro, Stefan; Gruis, Nelleke A; Corrie, Pippa; Iyer, Vivek; Robles-Espinoza, Carla Daniela; Puig-Butille, Joan A; Puig, Susana; Adams, David J

    2017-05-01

    Melanoma in young children is rare; however, its incidence in adolescents and young adults is rising. We describe the clinical course of a 15-year-old female diagnosed with AJCC stage IB non-ulcerated primary melanoma, who died from metastatic disease 4 years after diagnosis despite three lines of modern systemic therapy. We also present the complete genomic profile of her tumour and compare this to a further series of 13 adolescent melanomas and 275 adult cutaneous melanomas. A somatic BRAF V 600E mutation and a high mutational load equivalent to that found in adult melanoma and composed primarily of C>T mutations were observed. A germline genomic analysis alongside a series of 23 children and adolescents with melanoma revealed no mutations in known germline melanoma-predisposing genes. Adolescent melanomas appear to have genomes that are as complex as those arising in adulthood and their clinical course can, as with adults, be unpredictable. © 2017 The Authors. Pigment Cell & Melanoma Research published by John Wiley & Sons Ltd.

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

    PubMed Central

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

    2014-01-01

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

  13. A Systems Biology Approach Identifies FUT8 as a Driver of Melanoma Metastasis.

    PubMed

    Agrawal, Praveen; Fontanals-Cirera, Barbara; Sokolova, Elena; Jacob, Samson; Vaiana, Christopher A; Argibay, Diana; Davalos, Veronica; McDermott, Meagan; Nayak, Shruti; Darvishian, Farbod; Castillo, Mireia; Ueberheide, Beatrix; Osman, Iman; Fenyö, David; Mahal, Lara K; Hernando, Eva

    2017-06-12

    Association of aberrant glycosylation with melanoma progression is based mainly on analyses of cell lines. Here we present a systems-based study of glycomic changes and corresponding enzymes associated with melanoma metastasis in patient samples. Upregulation of core fucosylation (FUT8) and downregulation of α-1,2 fucosylation (FUT1, FUT2) were identified as features of metastatic melanoma. Using both in vitro and in vivo studies, we demonstrate FUT8 is a driver of melanoma metastasis which, when silenced, suppresses invasion and tumor dissemination. Glycoprotein targets of FUT8 were enriched in cell migration proteins including the adhesion molecule L1CAM. Core fucosylation impacted L1CAM cleavage and the ability of L1CAM to support melanoma invasion. FUT8 and its targets represent therapeutic targets in melanoma metastasis. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Recombinant Interleukin-15 in Treating Patients With Advanced Melanoma, Kidney Cancer, Non-small Cell Lung Cancer, or Squamous Cell Head and Neck Cancer

    ClinicalTrials.gov

    2017-09-14

    Head and Neck Squamous Cell Carcinoma; Recurrent Head and Neck Carcinoma; Recurrent Non-Small Cell Lung Carcinoma; Recurrent Renal Cell Carcinoma; Recurrent Skin Carcinoma; Stage III Renal Cell Cancer; Stage IIIA Cutaneous Melanoma AJCC v7; Stage IIIA Non-Small Cell Lung Cancer AJCC v7; Stage IIIB Cutaneous Melanoma AJCC v7; Stage IIIB Non-Small Cell Lung Cancer AJCC v7; Stage IIIC Cutaneous Melanoma AJCC v7; Stage IV Cutaneous Melanoma AJCC v6 and v7; Stage IV Non-Small Cell Lung Cancer AJCC v7; Stage IV Renal Cell Cancer

  15. The sodium pump α1 sub-unit: a disease progression–related target for metastatic melanoma treatment

    PubMed Central

    Mathieu, Véronique; Pirker, Christine; Martin de Lassalle, Elisabeth; Vernier, Mathieu; Mijatovic, Tatjana; DeNeve, Nancy; Gaussin, Jean-François; Dehoux, Mischael; Lefranc, Florence; Berger, Walter; Kiss, Robert

    2009-01-01

    Melanomas remain associated with dismal prognosis because they are naturally resistant to apoptosis and they markedly metastasize. Up-regulated expression of sodium pump α sub-units has previously been demonstrated when comparing metastatic to non-metastatic melanomas. Our previous data revealed that impairing sodium pump α1 activity by means of selective ligands, that are cardiotonic steroids, markedly impairs cell migration and kills apoptosis-resistant cancer cells. The objective of this study was to determine the expression levels of sodium pump α sub-units in melanoma clinical samples and cell lines and also to characterize the role of α1 sub-units in melanoma cell biology. Quantitative RT-PCR, Western blotting and immunohistochemistry were used to determine the expression levels of sodium pump α sub-units. In vitro cytotoxicity of various cardenolides and of an anti-α1 siRNA was evaluated by means of MTT assay, quantitative videomicroscopy and through apoptosis assays. The in vivo activity of a novel cardenolide UNBS1450 was evaluated in a melanoma brain metastasis model. Our data show that all investigated human melanoma cell lines expressed high levels of the α1 sub-unit, and 33% of human melanomas displayed significant α1 sub-unit expression in correlation with the Breslow index. Furthermore, cardenolides (notably UNBS1450; currently in Phase I clinical trials) displayed marked anti-tumour effects against melanomas in vitro. This activity was closely paralleled by decreases in cMyc expression and by increases in apoptotic features. UNBS1450 also displayed marked anti-tumour activity in the aggressive human metastatic brain melanoma model in vivo. The α1 sodium pump sub-unit could represent a potential novel target for combating melanoma. PMID:19243476

  16. UV-induced somatic mutations elicit a functional T cell response in the YUMMER1.7 mouse melanoma model.

    PubMed

    Wang, Jake; Perry, Curtis J; Meeth, Katrina; Thakral, Durga; Damsky, William; Micevic, Goran; Kaech, Susan; Blenman, Kim; Bosenberg, Marcus

    2017-07-01

    Human melanomas exhibit relatively high somatic mutation burden compared to other malignancies. These somatic mutations may produce neoantigens that are recognized by the immune system, leading to an antitumor response. By irradiating a parental mouse melanoma cell line carrying three driver mutations with UVB and expanding a single-cell clone, we generated a mutagenized model that exhibits high somatic mutation burden. When inoculated at low cell numbers in immunocompetent C57BL/6J mice, YUMMER1.7 (Yale University Mouse Melanoma Exposed to Radiation) regresses after a brief period of growth. This regression phenotype is dependent on T cells as YUMMER1.7 tumors grow significantly faster in immunodeficient Rag1 -/- mice and C57BL/6J mice depleted of CD4 and CD8 T cells. Interestingly, regression can be overcome by injecting higher cell numbers of YUMMER1.7, which results in tumors that grow without effective rejection. Mice that have previously rejected YUMMER1.7 tumors develop immunity against higher doses of YUMMER1.7 tumor challenge. In addition, escaping YUMMER1.7 tumors are sensitive to anti-CTLA-4 and anti-PD-1 therapy, establishing a new model for the evaluation of immune checkpoint inhibition and antitumor immune responses. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. A novel ATM-dependent checkpoint defect distinct from loss of function mutation promotes genomic instability in melanoma.

    PubMed

    Spoerri, Loredana; Brooks, Kelly; Chia, KeeMing; Grossman, Gavriel; Ellis, Jonathan J; Dahmer-Heath, Mareike; Škalamera, Dubravka; Pavey, Sandra; Burmeister, Bryan; Gabrielli, Brian

    2016-05-01

    Melanomas have high levels of genomic instability that can contribute to poor disease prognosis. Here, we report a novel defect of the ATM-dependent cell cycle checkpoint in melanoma cell lines that promotes genomic instability. In defective cells, ATM signalling to CHK2 is intact, but the cells are unable to maintain the cell cycle arrest due to elevated PLK1 driving recovery from the arrest. Reducing PLK1 activity recovered the ATM-dependent checkpoint arrest, and over-expressing PLK1 was sufficient to overcome the checkpoint arrest and increase genomic instability. Loss of the ATM-dependent checkpoint did not affect sensitivity to ionizing radiation demonstrating that this defect is distinct from ATM loss of function mutations. The checkpoint defective melanoma cell lines over-express PLK1, and a significant proportion of melanomas have high levels of PLK1 over-expression suggesting this defect is a common feature of melanomas. The inability of ATM to impose a cell cycle arrest in response to DNA damage increases genomic instability. This work also suggests that the ATM-dependent checkpoint arrest is likely to be defective in a higher proportion of cancers than previously expected. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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

    PubMed

    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.

  19. A novel immune resistance mechanism of melanoma cells controlled by the ADAR1 enzyme

    PubMed Central

    Galore-Haskel, Gilli; Nemlich, Yael; Greenberg, Eyal; Ashkenazi, Shira; Hakim, Motti; Itzhaki, Orit; Shoshani, Noa; Shapira-Fromer, Ronnie; Ben-Ami, Eytan; Ofek, Efrat; Anafi, Liat; Besser, Michal J.

    2015-01-01

    The blossom of immunotherapy in melanoma highlights the need to delineate mechanisms of immune resistance. Recently, we have demonstrated that the RNA editing protein, adenosine deaminase acting on RNA-1 (ADAR1) is down-regulated during metastatic transition of melanoma, which enhances melanoma cell proliferation and tumorigenicity. Here we investigate the role of ADAR1 in melanoma immune resistance. Importantly, knockdown of ADAR1 in human melanoma cells induces resistance to tumor infiltrating lymphocytes in a cell contact-dependent mechanism. We show that ADAR1, in an editing-independent manner, regulates the biogenesis of miR-222 at the transcription level and thereby Intercellular Adhesion Molecule 1 (ICAM1) expression, which consequently affects melanoma immune resistance. ADAR1 thus has a novel, pivotal, role in cancer immune resistance. Corroborating with these results, the expression of miR-222 in melanoma tissue specimens was significantly higher in patients who had no clinical benefit from treatment with ipilimumab as compared to patients that responded clinically, suggesting that miR-222 could function as a biomarker for the prediction of response to ipilimumab. These results provide not only novel insights on melanoma immune resistance, but also pave the way to the development of innovative personalized tools to enable optimal drug selection and treatment. PMID:26338962

  20. Plasma Membrane Integrity and Survival of Melanoma Cells After Nanosecond Laser Pulses

    PubMed Central

    Pérez-Gutiérrez, Francisco G.; Camacho-López, Santiago; Evans, Rodger; Guillén, Gabriel; Goldschmidt, Benjamin S.; Viator, John A.

    2010-01-01

    Circulating tumor cells (CTCs) photoacoustic detection systems can aid clinical decision-making in the treatment of cancer. Interaction of melanin within melanoma cells with nanosecond laser pulses generates photoacoustic waves that make its detection possible. This study aims at: (1) determining melanoma cell survival after laser pulses of 6 ns at λ = 355 and 532 nm; (2) comparing the potential enhancement in the photoacoustic signal using λ = 355 nm in contrast with λ = 532 nm; (3) determining the critical laser fluence at which melanin begins to leak out from melanoma cells; and (4) developing a time-resolved imaging (TRI) system to study the intracellular interactions and their effect on the plasma membrane integrity. Monolayers of melanoma cells were grown on tissue culture-treated clusters and irradiated with up to 1.0 J/cm2. Surviving cells were stained with trypan blue and counted using a hemacytometer. The phosphate buffered saline absorbance was measured with a nanodrop spectrophotometer to detect melanin leakage from the melanoma cells post-laser irradiation. Photoacoustic signal magnitude was studied at both wavelengths using piezoelectric sensors. TRI with 6 ns resolution was used to image plasma membrane damage. Cell survival decreased proportionally with increasing laser fluence for both wavelengths, although the decrease is more pronounced for 355 nm radiation than for 532 nm. It was found that melanin leaks from cells equally for both wavelengths. No significant difference in photoacoustic signal was found between wavelengths. TRI showed clear damage to plasma membrane due to laser-induced bubble formation. PMID:20589533

  1. Study of melanoma invasion by FTIR spectroscopy

    NASA Astrophysics Data System (ADS)

    Yang, Y.; Sulé-Suso, J.; Sockalingum, G. D.

    2008-02-01

    Compared to other forms of skin cancer, a malignant melanoma has a high risk of spreading to other parts of the body. Melanoma invasion is a complex process involving changes in cell-extracellular matrix (ECM) interaction and cell-cell interactions. To fully understand the factors which control the invasion process, a human skin model system was reconstructed. HBL (a commercially available cell line) melanoma cells were seeded on a skin model with and without the presence of keratinocytes and/or fibroblasts. After 14 days culture, the skin specimens were fixed, parafin embedded and cut into 7 µm sections. The de-parafinised sections were investigated by synchrotron Fourier transformed infrared (FTIR) microspectroscopy to study skin cell invasion behaviour. The advantage of using FTIR is its ability to obtain the fingerprint information of the invading cells in terms of protein secondary structure in comparison to non-invading cells and the concentration of the enzyme (matrix-metalloproteinase) which digests protein matrix, near the invading cells. With aid of the spectral mapping images, it is possible to pinpoint the cells in non-invasion and invasion area and analyse the respective spectra. It has been observed that the protein bands in cells and matrix shifted between non-invasive and invasive cells in the reconstructed skin model. We hypothesise that by careful analysis of the FTIR data and validation by other models, FTIR studies can reveal information on which type of cells and proteins are involved in melanoma invasion. Thus, it is possible to trace the cell invasion path by mapping the spectra along the interface of cell layer and matrix body by FTIR spectroscopy.

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

    PubMed

    Kushiro, Kyoko; Núñez, Nomelí P

    2012-01-01

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

  3. TIL therapy broadens the tumor-reactive CD8+ T cell compartment in melanoma patients

    PubMed Central

    Kvistborg, Pia; Shu, Chengyi Jenny; Heemskerk, Bianca; Fankhauser, Manuel; Thrue, Charlotte Albæk; Toebes, Mireille; van Rooij, Nienke; Linnemann, Carsten; van Buuren, Marit M.; Urbanus, Jos H.M.; Beltman, Joost B.; thor Straten, Per; Li, Yong F.; Robbins, Paul F.; Besser, Michal J.; Schachter, Jacob; Kenter, Gemma G.; Dudley, Mark E.; Rosenberg, Steven A.; Haanen, John B.A.G.; Hadrup, Sine Reker; Schumacher, Ton N.M.

    2012-01-01

    There is strong evidence that both adoptive T cell transfer and T cell checkpoint blockade can lead to regression of human melanoma. However, little data are available on the effect of these cancer therapies on the tumor-reactive T cell compartment. To address this issue we have profiled therapy-induced T cell reactivity against a panel of 145 melanoma-associated CD8+ T cell epitopes. Using this approach, we demonstrate that individual tumor-infiltrating lymphocyte cell products from melanoma patients contain unique patterns of reactivity against shared melanoma-associated antigens, and that the combined magnitude of these responses is surprisingly low. Importantly, TIL therapy increases the breadth of the tumor-reactive T cell compartment in vivo, and T cell reactivity observed post-therapy can almost in full be explained by the reactivity observed within the matched cell product. These results establish the value of high-throughput monitoring for the analysis of immuno-active therapeutics and suggest that the clinical efficacy of TIL therapy can be enhanced by the preparation of more defined tumor-reactive T cell products. PMID:22754759

  4. TIL therapy broadens the tumor-reactive CD8(+) T cell compartment in melanoma patients.

    PubMed

    Kvistborg, Pia; Shu, Chengyi Jenny; Heemskerk, Bianca; Fankhauser, Manuel; Thrue, Charlotte Albæk; Toebes, Mireille; van Rooij, Nienke; Linnemann, Carsten; van Buuren, Marit M; Urbanus, Jos H M; Beltman, Joost B; Thor Straten, Per; Li, Yong F; Robbins, Paul F; Besser, Michal J; Schachter, Jacob; Kenter, Gemma G; Dudley, Mark E; Rosenberg, Steven A; Haanen, John B A G; Hadrup, Sine Reker; Schumacher, Ton N M

    2012-07-01

    There is strong evidence that both adoptive T cell transfer and T cell checkpoint blockade can lead to regression of human melanoma. However, little data are available on the effect of these cancer therapies on the tumor-reactive T cell compartment. To address this issue we have profiled therapy-induced T cell reactivity against a panel of 145 melanoma-associated CD8(+) T cell epitopes. Using this approach, we demonstrate that individual tumor-infiltrating lymphocyte cell products from melanoma patients contain unique patterns of reactivity against shared melanoma-associated antigens, and that the combined magnitude of these responses is surprisingly low. Importantly, TIL therapy increases the breadth of the tumor-reactive T cell compartment in vivo, and T cell reactivity observed post-therapy can almost in full be explained by the reactivity observed within the matched cell product. These results establish the value of high-throughput monitoring for the analysis of immuno-active therapeutics and suggest that the clinical efficacy of TIL therapy can be enhanced by the preparation of more defined tumor-reactive T cell products.

  5. Loss of NF1 in cutaneous melanoma is associated with RAS activation and MEK dependence.

    PubMed

    Nissan, Moriah H; Pratilas, Christine A; Jones, Alexis M; Ramirez, Ricardo; Won, Helen; Liu, Cailian; Tiwari, Shakuntala; Kong, Li; Hanrahan, Aphrothiti J; Yao, Zhan; Merghoub, Taha; Ribas, Antoni; Chapman, Paul B; Yaeger, Rona; Taylor, Barry S; Schultz, Nikolaus; Berger, Michael F; Rosen, Neal; Solit, David B

    2014-04-15

    Melanoma is a disease characterized by lesions that activate ERK. Although 70% of cutaneous melanomas harbor activating mutations in the BRAF and NRAS genes, the alterations that drive tumor progression in the remaining 30% are largely undefined. Vemurafenib, a selective inhibitor of RAF kinases, has clinical utility restricted to BRAF-mutant tumors. MEK inhibitors, which have shown clinical activity in NRAS-mutant melanoma, may be effective in other ERK pathway-dependent settings. Here, we investigated a panel of melanoma cell lines wild type for BRAF and NRAS to determine the genetic alteration driving their transformation and their dependence on ERK signaling in order to elucidate a candidate set for MEK inhibitor treatment. A cohort of the BRAF/RAS wild type cell lines with high levels of RAS-GTP had loss of NF1, a RAS GTPase activating protein. In these cell lines, the MEK inhibitor PD0325901 inhibited ERK phosphorylation, but also relieved feedback inhibition of RAS, resulting in induction of pMEK and a rapid rebound in ERK signaling. In contrast, the MEK inhibitor trametinib impaired the adaptive response of cells to ERK inhibition, leading to sustained suppression of ERK signaling and significant antitumor effects. Notably, alterations in NF1 frequently co-occurred with RAS and BRAF alterations in melanoma. In the setting of BRAF(V600E), NF1 loss abrogated negative feedback on RAS activation, resulting in elevated activation of RAS-GTP and resistance to RAF, but not MEK, inhibitors. We conclude that loss of NF1 is common in cutaneous melanoma and is associated with RAS activation, MEK-dependence, and resistance to RAF inhibition. ©2014 AACR.

  6. pH-dependent antitumor activity of proton pump inhibitors against human melanoma is mediated by inhibition of tumor acidity.

    PubMed

    De Milito, Angelo; Canese, Rossella; Marino, Maria Lucia; Borghi, Martina; Iero, Manuela; Villa, Antonello; Venturi, Giulietta; Lozupone, Francesco; Iessi, Elisabetta; Logozzi, Mariantonia; Della Mina, Pamela; Santinami, Mario; Rodolfo, Monica; Podo, Franca; Rivoltini, Licia; Fais, Stefano

    2010-07-01

    Metastatic melanoma is associated with poor prognosis and still limited therapeutic options. An innovative treatment approach for this disease is represented by targeting acidosis, a feature characterizing tumor microenvironment and playing an important role in cancer malignancy. Proton pump inhibitors (PPI), such as esomeprazole (ESOM) are prodrugs functionally activated by acidic environment, fostering pH neutralization by inhibiting proton extrusion. We used human melanoma cell lines and xeno-transplated SCID mice to provide preclinical evidence of ESOM antineoplastic activity. Human melanoma cell lines, characterized by different mutation and signaling profiles, were treated with ESOM in different pH conditions and evaluated for proliferation, viability and cell death. SCID mice engrafted with human melanoma were used to study ESOM administration effects on tumor growth and tumor pH by magnetic resonance spectroscopy (MRS). ESOM inhibited proliferation of melanoma cells in vitro and induced a cytotoxicity strongly boosted by low pH culture conditions. ESOM-induced tumor cell death occurred via rapid intracellular acidification and activation of several caspases. Inhibition of caspases activity by pan-caspase inhibitor z-vad-fmk completely abrogated the ESOM-induced cell death. ESOM administration (2.5 mg kg(-1)) to SCID mice engrafted with human melanoma reduced tumor growth, consistent with decrease of proliferating cells and clear reduction of pH gradients in tumor tissue. Moreover, systemic ESOM administration dramatically increased survival of human melanoma-bearing animals, in absence of any relevant toxicity. These data show preclinical evidence supporting the use of PPI as novel therapeutic strategy for melanoma, providing the proof of concept that PPI target human melanoma modifying tumor pH gradients.

  7. Anticancer potential of benzothiazolic derivative (E)-2-((2-(benzo[d]thiazol-2-yl)hydrazono)methyl)-4-nitrophenol against melanoma cells.

    PubMed

    Vasconcelos, Zanair Soares; Ralph, Ana Carolina Lima; Calcagno, Danielle Queiroz; Dos Santos Barbosa, Gleyce; do Nascimento Pedrosa, Tatiana; Antony, Lucas Pio; de Arruda Cardoso Smith, Marília; de Lucas Chazin, Eliza; Vasconcelos, Thatyana Rocha Alves; Montenegro, Raquel Carvalho; de Vasconcellos, Marne Carvalho

    2018-08-01

    Malignant melanoma is an important type of cancer worldwide due to its aggressiveness and poor survival rate. Significant efforts to understand the biology of melanoma and approaches to treat the advanced disease are focused on targeted gene inhibitors. Frequently mutated genes, such as NRAS, B-RAF and TP53, significantly exceed the frequency of mutations of other genes, emphasizing their importance for future targeted therapies. Considering the antitumor activity of benzothiazolic derivatives, this study aimed to demonstrate the action of benzothiazolic (E)-2-((2-(benzo[d]thiazol-2-yl)hydrazono)methyl)-4-nitrophenol (AFN01) against three established human melanoma cell lines that recapitulate the molecular landscape of the disease in terms of its genetic alterations and mutations, such as the TP53, NRAS and B-RAF genes. The results presented here indicate that AFN01, as a significant cytostatic and cytotoxic drug due to its induction of DNA fragmentation, causes single and double DNA strand breaks, consequently inhibiting cell proliferation, migration and invasion by promoting apoptosis. Our data suggest that AFN01 might be considered as a future therapeutic option for managing melanoma. Copyright © 2018 Elsevier Ltd. All rights reserved.

  8. NGF reprograms metastatic melanoma to a bipotent glial-melanocyte neural crest-like precursor

    PubMed Central

    Kasemeier-Kulesa, Jennifer C.; Romine, Morgan H.; Morrison, Jason A.; Bailey, Caleb M.; Welch, Danny R.

    2018-01-01

    ABSTRACT Melanoma pathogenesis from normal neural crest-derived melanocytes is often fatal due to aggressive cell invasion throughout the body. The identification of signals that reprogram de-differentiated, metastatic melanoma cells to a less aggressive and stable phenotype would provide a novel strategy to limit disease progression. In this study, we identify and test the function of developmental signals within the chick embryonic neural crest microenvironment to reprogram and sustain the transition of human metastatic melanoma to a neural crest cell-like phenotype. Results reveal that co-culture of the highly aggressive and metastatic human melanoma cell line C8161 upregulate a marker of melanosome formation (Mart-1) in the presence of embryonic day 3.5 chick trunk dorsal root ganglia. We identify nerve growth factor (NGF) as the signal within this tissue driving Mart-1 re-expression and show that NGF receptors trkA and p75 cooperate to induce Mart-1 re-expression. Furthermore, Mart-1 expressing C8161 cells acquire a gene signature of poorly aggressive C81-61 cells. These data suggest that targeting NGF signaling may yield a novel strategy to reprogram metastatic melanoma toward a benign cell type. PMID:29175861

  9. Characterizations of 9p21 candidate genes in familial melanoma

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

    Walker, G.J.; Flores, J.F.; Glendening, J.M.

    We have previously collected and characterized 16 melanoma families for the inheritance of a familial melanoma predisposition gene on 9p21. Clear evidence for genetic linkage has been detected in 8 of these families with the 9p21 markers D9S126 and 1FNA, while linkage of the remaining families to this region is less certain. A candidate for the 9p21 familial melanoma gene, the cyclin kinase inhibitor gene p16 (also known as the multiple tumor suppressor 1 (MTS1) gene), has been recently indentified. Notably, a nonsense mutation within the p16 gene has been detected in the lymphoblastoid cell line DNA from a dysplasticmore » nevus syndrome (DNS), or familial melanoma, patient. The p16 gene is also known to be frequently deleted or mutated in a variety of tumor cell lines (including melanoma) and resides within a region that has been defined as harboring the 9p21 melanoma predisposition locus. This region is delineated on the distal side by the marker D9S736 (which resides just distal to the p16 gene) and extends in a proximal direction to the marker D9S171. Overall, the entire distance between these two loci is estimated at 3-5Mb. Preliminary analysis of our two largest 9p21-linked melanoma kindreds (by direct sequencing of PCR products) has not yet revealed mutations within the coding region of the p16 gene. Others have reported that 8/11 unrelated 9p21-linked melanoma families do not appear to carry p16 mutations; thus the possibility exists that p16 is not a melanoma susceptibility gene per se, although it appears to play some role in melanoma tumor progression. Our melanoma kindred DNAs are currently being analyzed by SSCP using primers that amplify exons of other candidate genes from the 9p21 region implicated in familial melanoma. These novel genes reside within a distinct critical region of homozygous loss in melanoma which is located >2 Mb from the p16 gene on 9p21.« less

  10. Reprogramming of Melanoma Tumor-Infiltrating Lymphocytes to Induced Pluripotent Stem Cells

    PubMed Central

    Saito, Hidehito; Okita, Keisuke; Fusaki, Noemi; Sabel, Michael S.; Chang, Alfred E.; Ito, Fumito

    2016-01-01

    Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients hold great promise for autologous cell therapies. One of the possible applications of iPSCs is to use them as a cell source for producing autologous lymphocytes for cell-based therapy against cancer. Tumor-infiltrating lymphocytes (TILs) that express programmed cell death protein-1 (PD-1) are tumor-reactive T cells, and adoptive cell therapy with autologous TILs has been found to achieve durable complete response in selected patients with metastatic melanoma. Here, we describe the derivation of human iPSCs from melanoma TILs expressing high level of PD-1 by Sendai virus-mediated transduction of the four transcription factors, OCT3/4, SOX2, KLF4, and c-MYC. TIL-derived iPSCs display embryonic stem cell-like morphology, have normal karyotype, express stem cell-specific surface antigens and pluripotency-associated transcription factors, and have the capacity to differentiate in vitro and in vivo. A wide variety of T cell receptor gene rearrangement patterns in TIL-derived iPSCs confirmed the heterogeneity of T cells infiltrating melanomas. The ability to reprogram TILs containing patient-specific tumor-reactive repertoire might allow the generation of patient- and tumor-specific polyclonal T cells for cancer immunotherapy. PMID:27057178

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2012-03-01

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

  13. Anti-proliferative activity of 2,6-dichloro-9- or 7-(ethoxycarbonylmethyl)-9H- or 7H-purines against several human solid tumour cell lines.

    PubMed

    Morales, Fátima; Ramírez, Alberto; Conejo-García, Ana; Morata, Cynthia; Marchal, Juan A; Campos, Joaquín M

    2014-04-09

    As leads we took several benzo-fused seven- and six-membered scaffolds linked to the pyrimidine or purine moieties with notable anti-proliferative activity against human breast, colon and melanoma cancerous cell lines. We then decided to maintain the double-ringed nitrogenous bases and change the other components to the ethyl acetate moiety. This way six purine and two 5-fluorouracil derivatives were obtained and evaluated against the MCF-7, HCT-116, A-375 and G-361 cancer cell lines. Two QSARs are obtained between the anti-proliferative IC₅₀ values for compounds 26-33 and the clog P against the melanoma cell lines A-375 and G-361. Our results show that two of the analogues [ethyl 2-(2,6-dichloro-9H- or 7H-purine-9- or 7-yl)acetates (30 and 33, respectively)] are potent cytotoxic agents against all the tumour cell lines assayed, showing single-digit micromolar IC₅₀ values. This exemplifies the potential of our previously reported purine compounds to qualify as lead structures for medicinal chemistry campaigns, affording simplified analogues easy to synthesize and with a noteworthy bioactivity. The selective activity of 30 and 33 against the melanoma cell line A-375, via apoptosis, supposes a great advantage for a future therapeutic use. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  14. Direct targeting of MEK1/2 and RSK2 by silybin induces cell cycle arrest and inhibits melanoma cell growth

    PubMed Central

    Lee, Mee-Hyun; Huang, Zunnan; Kim, Dong Joon; Kim, Sung-Hyun; Kim, Myoung Ok; Lee, Sung-Young; Xie, Hua; Park, Si Jun; Kim, Jae Young; Kundu, Joydeb Kumar; Bode, Ann M.; Surh, Young-Joon; Dong, Zigang

    2013-01-01

    Abnormal functioning of multiple gene products underlies the neoplastic transformation of cells. Thus, chemopreventive and/or chemotherapeutic agents with multigene targets hold promise in the development of effective anticancer drugs. Silybin, a component of milk thistle, is a natural anticancer agent. In the present study, we investigated the effect of silybin on melanoma cell growth and elucidated its molecular targets. Our study revealed that silybin attenuated the growth of melanoma xenograft tumors in nude mice. Silybin inhibited the kinase activity of mitogen-activated protein kinase kinase (MEK)-1/2 and ribosomal S6 kinase (RSK)-2 in melanoma cells. The direct binding of silybin with MEK1/2 and RSK2 was explored using a computational docking model. Treatment of melanoma cells with silybin attenuated the phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 and RSK2, which are regulated by the upstream kinases MEK1/2. The blockade of MEK1/2-ERK1/2-RSK2 signaling by silybin resulted in a reduced activation of nuclear factor-kappaB, activator protein-1 and signal transducer and activator of transcription-3, which are transcriptional regulators of a variety of proliferative genes in melanomas. Silybin, by blocking the activation of these transcription factors, induced cell cycle arrest at the G1 phase and inhibited melanoma cell growth in vitro and in vivo. Taken together, silybin suppresses melanoma growth by directly targeting MEK- and RSK-mediated signaling pathways. PMID:23447564

  15. Eradication of melanoma in vitro and in vivo via targeting with a Killer-Red-containing telomerase-dependent adenovirus.

    PubMed

    Takehara, Kiyoto; Yano, Shuya; Tazawa, Hiroshi; Kishimoto, Hiroyuki; Narii, Nobuhiro; Mizuguchi, Hiroyuki; Urata, Yasuo; Kagawa, Shunsuke; Fujiwara, Toshiyoshi; Hoffman, Robert M

    2017-08-18

    Melanoma is a highly recalcitrant cancer and transformative therapy is necessary for the cure of this disease. We recently developed a telomerase-dependent adenovirus containing the fluorescent protein Killer-Red. In the present report, we first determined the efficacy of Killer-Red adenovirus combined with laser irradiation on human melanoma cell lines in vitro. Cell viability of human melanoma cells was reduced in a dose-dependent and irradiation-time-dependent manner. We used an intradermal xenografted melanoma model in nude mice to determine efficacy of the Killer-Red adenovirus. Intratumoral injection of Killer-Red adenovirus, combined with laser irradiation, eradicated the melanoma indicating the potential of a new paradigm of cancer therapy.

  16. Silencing FLI or targeting CD13/ANPEP lead to dephosphorylation of EPHA2, a mediator of BRAF inhibitor resistance, and induce growth arrest or apoptosis in melanoma cells.

    PubMed

    Azimi, Alireza; Tuominen, Rainer; Costa Svedman, Fernanda; Caramuta, Stefano; Pernemalm, Maria; Frostvik Stolt, Marianne; Kanter, Lena; Kharaziha, Pedram; Lehtiö, Janne; Hertzman Johansson, Carolina; Höiom, Veronica; Hansson, Johan; Egyhazi Brage, Suzanne

    2017-08-31

    A majority of patients with BRAF-mutated metastatic melanoma respond to therapy with BRAF inhibitors (BRAFi), but relapses are common owing to acquired resistance. To unravel BRAFi resistance mechanisms we have performed gene expression and mass spectrometry based proteome profiling of the sensitive parental A375 BRAF V600E-mutated human melanoma cell line and of daughter cell lines with induced BRAFi resistance. Increased expression of two novel resistance candidates, aminopeptidase-N (CD13/ANPEP) and ETS transcription factor FLI1 was observed in the BRAFi-resistant daughter cell lines. In addition, increased levels of the previously reported resistance mediators, receptor tyrosine kinase ephrine receptor A2 (EPHA2) and the hepatocyte growth factor receptor MET were also identified. The expression of these proteins was assessed in matched tumor samples from melanoma patients obtained before BRAFi and after disease progression. MET was overexpressed in all progression samples while the expression of the other candidates varied between the individual patients. Targeting CD13/ANPEP by a blocking antibody induced apoptosis in both parental A375- and BRAFi-resistant daughter cells as well as in melanoma cells with intrinsic BRAFi resistance and led to dephosphorylation of EPHA2 on S897, previously demonstrated to cause inhibition of the migratory capacity. AKT and RSK, both reported to induce EPHA2 S897 phosphorylation, were also dephosphorylated after inhibition of CD13/ANPEP. FLI1 silencing also caused decreases in EPHA2 S897 phosphorylation and in total MET protein expression. In addition, silencing of FLI1 sensitized the resistant cells to BRAFi. Furthermore, we show that BRAFi in combination with the multi kinase inhibitor dasatinib can abrogate BRAFi resistance and decrease both EPHA2 S897 phosphorylation and total FLI1 protein expression. This is the first report presenting CD13/ANPEP and FLI1 as important mediators of resistance to BRAF inhibition with

  17. Intercellular crosstalk in human malignant melanoma.

    PubMed

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

    2017-05-01

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

  18. Profiling of ABC transporters ABCB5, ABCF2 and nestin-positive stem cells in nevi, in situ and invasive melanoma.

    PubMed

    Setia, Namrata; Abbas, Ossama; Sousa, Yessica; Garb, Jane L; Mahalingam, Meera

    2012-08-01

    Distinct ABCB5 forms and ABCF2, members of the ATP-binding cassette (ABC) superfamily of transporters, are normally expressed in various tissues and cells, and enhanced expression of both has been demonstrated in select cancers. In melanoma cell lines, gene expression profiling of ABC transporters has revealed enhanced expression of melanocyte-specific ABCB5 and ABCF2 proteins. Given this, our primary aim was to ascertain immunohistochemical expression of the ABC transporters ABCB5 and ABCF2 and, the stem cell marker, nestin in a spectrum of benign and malignant nevomelanocytic proliferations, including nevi (n=30), in situ (n=31) and invasive (n=24) primary cutaneous melanomas to assess their role in the stepwise development of malignancy. In addition, their expression was compared with established melanoma prognosticators to ascertain their utility as independent prognosticators. A semiquantitative scoring system was utilized by deriving a cumulative score (based on percentage positivity cells and intensity of expression) and statistical analyses was carried out using analysis of variance with linear contrasts. Mean cumulative score in nevi, in situ and invasive melanoma were as follows: 3.8, 4.4 and 5.3 for ABCB5, respectively (P<0.005 for all), and 4.6, 4.6 and 5.3 for nestin, respectively (P=not significant for all). No appreciable expression of ABCF2 was noted in any of the groups. While ulcerated lesions of melanoma demonstrated lower levels of expression of ABCB5 and nestin than non-ulcerated lesions, and nestin expression was lower in lesions with mitoses >1, after controlling for the presence of ulceration and mitotic activity, the expression of both proteins did not significantly correlate with known melanoma prognosticators. The gradual increase in the expression of ABCB5 from benign nevus to in situ to invasive melanoma suggests that it plays a role in melanomagenesis. On the basis of our findings, a prospective study with follow-up data is required to

  19. Monoclonal Antibody and an Antibody-Toxin Conjugate to a Cell Surface Proteoglycan of Melanoma Cells Suppress in vivo Tumor Growth

    NASA Astrophysics Data System (ADS)

    Bumol, T. F.; Wang, Q. C.; Reisfeld, R. A.; Kaplan, N. O.

    1983-01-01

    A monoclonal antibody directed against a cell surface chondroitin sulfate proteoglycan of human melanoma cells, 9.2.27, and its diphtheria toxin A chain (DTA) conjugate were investigated for their effects on in vitro protein synthesis and in vivo tumor growth of human melanoma cells. The 9.2.27 IgG and its DTA conjugate display similar serological activities against melanoma target cells but only the conjugate can induce consistent in vitro inhibition of protein synthesis and toxicity in M21 melanoma cells. However, both 9.2.27 IgG and its DTA conjugate effect significant suppression of M21 tumor growth in vivo in an immunotherapy model of a rapidly growing tumor in athymic nu/nu mice, suggesting that other host mechanisms may mediate monoclonal antibody-induced tumor suppression.

  20. Ligand-activated BMP signaling inhibits cell differentiation and death to promote melanoma

    PubMed Central

    Venkatesan, Arvind M.; Vyas, Rajesh; Gramann, Alec K.; Gujja, Sharvari; Bhatnagar, Sanchita; Gomes, Camilla Borges Ferreira; Xi, Hualin Simon; Lian, Christine G.; Houvras, Yariv; Edwards, Yvonne J. K.; Deng, April; Ceol, Craig J.

    2017-01-01

    Oncogenomic studies indicate that copy number variation (CNV) alters genes involved in tumor progression; however, identification of specific driver genes affected by CNV has been difficult, as these rearrangements are often contained in large chromosomal intervals among several bystander genes. Here, we addressed this problem and identified a CNV-targeted oncogene by performing comparative oncogenomics of human and zebrafish melanomas. We determined that the gene encoding growth differentiation factor 6 (GDF6), which is the ligand for the BMP family, is recurrently amplified and transcriptionally upregulated in melanoma. GDF6-induced BMP signaling maintained a trunk neural crest gene signature in melanomas. Additionally, GDF6 repressed the melanocyte differentiation gene MITF and the proapoptotic factor SOX9, thereby preventing differentiation, inhibiting cell death, and promoting tumor growth. GDF6 was specifically expressed in melanomas but not melanocytes. Moreover, GDF6 expression levels in melanomas were inversely correlated with patient survival. Our study has identified a fundamental role for GDF6 and BMP signaling in governing an embryonic cell gene signature to promote melanoma progression, thus providing potential opportunities for targeted therapy to treat GDF6-positive cancers. PMID:29202482

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

    PubMed

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

    2017-03-01

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

  2. Mapping heterogeneity in patient-derived melanoma cultures by single-cell RNA-seq

    PubMed Central

    Loeffler-Wirth, Henry; Hopp, Lydia; Schadendorf, Dirk; Schartl, Manfred; Anderegg, Ulf; Camp, Gray; Treutlein, Barbara; Binder, Hans; Kunz, Manfred

    2017-01-01

    Recent technological advances in single-cell genomics make it possible to analyze cellular heterogeneity of tumor samples. Here, we applied single-cell RNA-seq to measure the transcriptomes of 307 single cells cultured from three biopsies of three different patients with a BRAF/NRAS wild type, BRAF mutant/NRAS wild type and BRAF wild type/NRAS mutant melanoma metastasis, respectively. Analysis based on self-organizing maps identified sub-populations defined by multiple gene expression modules involved in proliferation, oxidative phosphorylation, pigmentation and cellular stroma. Gene expression modules had prognostic relevance when compared with gene expression data from published melanoma samples and patient survival data. We surveyed kinome expression patterns across sub-populations of the BRAF/NRAS wild type sample and found that CDK4 and CDK2 were consistently highly expressed in the majority of cells, suggesting that these kinases might be involved in melanoma progression. Treatment of cells with the CDK4 inhibitor palbociclib restricted cell proliferation to a similar, and in some cases greater, extent than MAPK inhibitors. Finally, we identified a low abundant sub-population in this sample that highly expressed a module containing ABC transporter ABCB5, surface markers CD271 and CD133, and multiple aldehyde dehydrogenases (ALDHs). Patient-derived cultures of the BRAF mutant/NRAS wild type and BRAF wild type/NRAS mutant metastases showed more homogeneous single-cell gene expression patterns with gene expression modules for proliferation and ABC transporters. Taken together, our results describe an intertumor and intratumor heterogeneity in melanoma short-term cultures which might be relevant for patient survival, and suggest promising targets for new treatment approaches in melanoma therapy. PMID:27903987

  3. AMPK activators inhibit the proliferation of human melanomas bearing the activated MAPK pathway.

    PubMed

    Petti, Carlotta; Vegetti, Claudia; Molla, Alessandra; Bersani, Ilaria; Cleris, Loredana; Mustard, Kirsty J; Formelli, Franca; Hardie, Grahame D; Sensi, Marialuisa; Anichini, Andrea

    2012-10-01

    Raf/MEK/ERK signaling can inhibit the liver kinase B1-AMP-activated protein kinase (LKB1-AMPK) pathway, thus rendering melanoma cells resistant to energy stress conditions. We evaluated whether pharmacological reactivation of the AMPK function could exert antitumor effects on melanoma cells bearing this pathway constitutively active because of a mutation in NRAS or BRAF genes. Nine melanoma cell lines were treated with the AMPK activators 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR) and phenformin. The activation of AMPK enzymatic activity, phosphorylation of AMPK and acetyl-CoA carboxylase kinase, in-vitro proliferation, cell cycle, and in-vivo growth of xenografts in nude mice were evaluated. AICAR and phenformin promoted phosphorylation and enzymatic activity of AMPK, as well as phosphorylation of the AMPK downstream target acetyl-CoA carboxylase. Drug treatment of either BRAF-mutant or NRAS-mutant melanomas, at doses not inducing cell death, was accompanied by a dose-dependent decrease in melanoma cell proliferation because of cell cycle arrest in either the G0/G1 or the S phase, associated with an increased expression of the p21 cell cycle inhibitor. Melanomas isolated from subcutaneously implanted mice, 25 days from treatment with AICAR, showed increased staining of the senescence-associated marker β-galactosidase, high p21 expression, and evidence of necrosis. Altogether, these results indicate that pharmacological activators of AMPK-dependent pathways inhibit the cell growth of melanoma cells with active Raf/MEK/ERK signaling and provide a rationale for further investigation on their use in combination therapies.

  4. Autologous cytokine-induced killer cell immunotherapy may improve overall survival in advanced malignant melanoma patients.

    PubMed

    Zhang, Yong; Zhu, Yu'nan; Zhao, Erjiang; He, Xiaolei; Zhao, Lingdi; Wang, Zibing; Fu, Xiaomin; Qi, Yalong; Ma, Baozhen; Song, Yongping; Gao, Quanli

    2017-11-01

    Our study was conducted to explore the efficacy of autologous cytokine-induced killer (CIK) cells in patients with advanced malignant melanoma. Materials & Methods: Here we reviewed 113 stage IV malignant melanoma patients among which 68 patients received CIK cell immunotherapy alone, while 45 patients accepted CIK cell therapy combined with chemotherapy. Results: We found that the median survival time in CIK cell group was longer than the combined therapy group (21 vs 15 months, p = 0.07). In addition, serum hemoglobin level as well as monocyte proportion and lymphocyte count were associated with patients' survival time. These indicated that CIK cell immunotherapy might extend survival time in advanced malignant melanoma patients. Furthermore, serum hemoglobin level, monocyte proportion and lymphocyte count could be prognostic indicators for melanoma.

  5. Melanoma cells revive an embryonic transcriptional network to dictate phenotypic heterogeneity.

    PubMed

    Vandamme, Niels; Berx, Geert

    2014-01-01

    Compared to the overwhelming amount of literature describing how epithelial-to-mesenchymal transition (EMT)-inducing transcription factors orchestrate cellular plasticity in embryogenesis and epithelial cells, the functions of these factors in non-epithelial contexts, such as melanoma, are less clear. Melanoma is an aggressive tumor arising from melanocytes, endowed with unique features of cellular plasticity. The reversible phenotype-switching between differentiated and invasive phenotypes is increasingly appreciated as a mechanism accounting for heterogeneity in melanoma and is driven by oncogenic signaling and environmental cues. This phenotypic switch is coupled with an intriguing and somewhat counterintuitive signaling switch of EMT-inducing transcription factors. In contrast to carcinomas, different EMT-inducing transcription factors have antagonizing effects in melanoma. Balancing between these different EMT transcription factors is likely the key to successful metastatic spread of melanoma.

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

    PubMed

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

    2018-04-10

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

  7. Ex vivo enrichment of circulating anti-tumor T cells from both cutaneous and ocular melanoma patients: clinical implications for adoptive cell transfer therapy.

    PubMed

    Mazzarella, Tonia; Cambiaghi, Valeria; Rizzo, Nathalie; Pilla, Lorenzo; Parolini, Danilo; Orsenigo, Elena; Colucci, Annalisa; Modorati, Giulio; Doglioni, Claudio; Parmiani, Giorgio; Maccalli, Cristina

    2012-08-01

    Tumor-infiltrating lymphocytes (TILs) have been successfully used for adoptive cell transfer (ACT) immunotherapy; however, due to their scarce availability, this therapy is possible for a limited fraction of cutaneous melanoma patients. We assessed whether an effective protocol for ex vivo T-cell expansion from peripheral blood mononuclear cells (PBMCs), suitable for ACT of both cutaneous and ocular melanoma patients, could be identified. PBMCs from both cutaneous and ocular melanoma patients were stimulated in vitro with autologous, irradiated melanoma cells (mixed lymphocyte tumor cell culture; MLTCs) in the presence of IL-2 and IL-15 followed by the rapid expansion protocol (REP). The functional activity of these T lymphocytes was characterized and compared with that of TILs. In addition, the immune infiltration in vivo of ocular melanoma lesions was analyzed. An efficient in vitro MLTC expansion of melanoma reactive T cells was achieved from all PBMC's samples obtained in 7 cutaneous and ocular metastatic melanoma patients. Large numbers of melanoma-specific T cells could be obtained when the REP protocol was applied to these MLTCs. Most MLTCs were enriched in non-terminally differentiated T(EM) cells homogeneously expressing co-stimulatory molecules (e.g., NKG2D, CD28, CD134, CD137). A similar pattern of anti-tumor activity, in association with a more variable expression of co-stimulatory molecules, was detected on short-term in vitro cultured TILs isolated from the same patients. In these ocular melanoma patients, we observed an immune infiltrate with suppressive characteristics and a low rate of ex vivo growing TILs (28.5% of our cases). Our MLTC protocol overcomes this limitation, allowing the isolation of T lymphocytes with effector functions even in these patients. Thus, anti-tumor circulating PBMC-derived T cells could be efficiently isolated from melanoma patients by our novel ex vivo enrichment protocol. This protocol appears suitable for ACT studies

  8. Expression of Tissue Factor by Melanoma Cells Promotes Efficient Hematogenous Metastasis

    NASA Astrophysics Data System (ADS)

    Mueller, Barbara M.; Reisfeld, Ralph A.; Edgington, Thomas S.; Ruf, Wolfram

    1992-12-01

    Metastasis is a multistep process which requires highly adapted interactions of tumor cells with host target organs. Compared with nonmetastatic cells, metastatic human melanoma cells express 1000-fold higher levels of tissue factor (TF), the major cellular initiator of the plasma coagulation protease cascades. To explore whether TF may contribute to metastatic tumor dissemination, we analyzed the effect of specific inhibition of TF function on human melanoma metastasis in severe combined immunodeficient (SCID) mice. Using species-specific antibodies to TF, we demonstrate that initial adherence is insufficient for successful tumor cell implantation in a target organ. Rapid arrest of human tumor cells in the lungs of mice was not diminished by inhibition of TF. However, inhibition of TF receptor function and consequent reduction in local protease generation abolished prolonged adherence of tumor cells, resulting in significantly reduced numbers of tumor cells retained in the vasculature of the lungs. The growth of pulmonary metastases was also significantly inhibited by a blocking anti-TF monoclonal antibody and Fab fragments thereof, whereas a noninhibitory antibody lacked antimetastatic effects. Cell surface expression of functional TF thus contributes to melanoma progression by allowing metastatic cells to provide requisite signals for prolonged adhesive interactions and/or transmigration of tumor cells across the endothelium, resulting in successful metastatic tumor implantation.

  9. Phenformin enhances the therapeutic benefit of BRAFV600E inhibition in melanoma

    PubMed Central

    Yuan, Ping; Ito, Koichi; Perez-Lorenzo, Rolando; Del Guzzo, Christina; Lee, Jung Hyun; Shen, Che-Hung; Bosenberg, Marcus W.; McMahon, Martin; Cantley, Lewis C.; Zheng, Bin

    2013-01-01

    Biguanides, such as the diabetes therapeutics metformin and phenformin, have demonstrated antitumor activity both in vitro and in vivo. The energy-sensing AMP-activated protein kinase (AMPK) is known to be a major cellular target of biguanides. Based on our discovery of cross-talk between the AMPK and v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) signaling pathways, we investigated the antitumor effects of combining phenformin with a BRAF inhibitor PLX4720 on the proliferation of BRAF-mutated melanoma cells in vitro and on BRAF-driven tumor growth in vivo. Cotreatment of BRAF-mutated melanoma cell lines with phenformin and PLX4720 resulted in synergistic inhibition of cell viability, compared with the effects of the single agent alone. Moreover, treatment with phenformin significantly delayed the development of resistance to PLX4720 in cultured melanoma cells. Biochemical analyses showed that phenformin and PLX4720 exerted cooperative effects on inhibiting mTOR signaling and inducing apoptosis. Noticeably, phenformin selectively targeted subpopulations of cells expressing JARID1B, a marker for slow cycling melanoma cells, whereas PLX4720 selectively targeted JARID1B-negative cells. Finally, in contrast to their use as single agents, the combination of phenformin and PLX4720 induced tumor regression in both nude mice bearing melanoma xenografts and in a genetically engineered BRAFV600E/PTENnull-driven mouse model of melanoma. These results strongly suggest that significant therapeutic advantage may be achieved by combining AMPK activators such as phenformin with BRAF inhbitors for the treatment of melanoma. PMID:24145418

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

    PubMed

    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.

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

    PubMed

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

    2014-08-01

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

  12. EMMPRIN regulates β1 integrin-mediated adhesion through Kindlin-3 in human melanoma cells.

    PubMed

    Delyon, Julie; Khayati, Farah; Djaafri, Ibtissem; Podgorniak, Marie-Pierre; Sadoux, Aurélie; Setterblad, Niclas; Boutalbi, Zineb; Maouche, Kamel; Maskos, Uwe; Menashi, Suzanne; Lebbé, Céleste; Mourah, Samia

    2015-06-01

    EMMPRIN is known to promote tumor invasion through extracellular matrix (ECM) degradation. Here we report that EMMPRIN can regulate melanoma cell adhesion to the ECM through an interaction with β1 integrin involving kindlin-3. In this study, EMMPRIN knockdown in the human melanoma cell line M10 using siRNA decreased cell invasion and significantly increased cell adhesion and spreading. A morphological change from a round to a spread shape was observed associated with enhanced phalloidin-labelled actin staining. In situ proximity ligation assay and co-immunoprecipitation revealed that EMMPRIN silencing increased the interaction of β1 integrin with kindlin-3, a focal adhesion protein. This was associated with an increase in β1 integrin activation and a decrease in the phosphorylation of the downstream integrin kinase FAK. Moreover, the expression at both the transcript and protein level of kindlin-3 and of β1 integrin was inversely regulated by EMMPRIN. EMMPRIN did not regulate either talin expression or its interaction with β1 integrin. These results are consistent with our in vivo demonstration that EMMPRIN inhibition increased β1 integrin activation and its interaction with kindlin-3. To conclude, these findings reveal a new role of EMMPRIN in tumor cell migration through ß1 integrin/kindlin-3-mediated adhesion pathway. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. Immune cell-poor melanomas benefit from PD-1 blockade after targeted type I IFN activation.

    PubMed

    Bald, Tobias; Landsberg, Jennifer; Lopez-Ramos, Dorys; Renn, Marcel; Glodde, Nicole; Jansen, Philipp; Gaffal, Evelyn; Steitz, Julia; Tolba, Rene; Kalinke, Ulrich; Limmer, Andreas; Jönsson, Göran; Hölzel, Michael; Tüting, Thomas

    2014-06-01

    Infiltration of human melanomas with cytotoxic immune cells correlates with spontaneous type I IFN activation and a favorable prognosis. Therapeutic blockade of immune-inhibitory receptors in patients with preexisting lymphocytic infiltrates prolongs survival, but new complementary strategies are needed to activate cellular antitumor immunity in immune cell-poor melanomas. Here, we show that primary melanomas in Hgf-Cdk4(R24C) mice, which imitate human immune cell-poor melanomas with a poor outcome, escape IFN-induced immune surveillance and editing. Peritumoral injections of immunostimulatory RNA initiated a cytotoxic inflammatory response in the tumor microenvironment and significantly impaired tumor growth. This critically required the coordinated induction of type I IFN responses by dendritic, myeloid, natural killer, and T cells. Importantly, antibody-mediated blockade of the IFN-induced immune-inhibitory interaction between PD-L1 and PD-1 receptors further prolonged the survival. These results highlight important interconnections between type I IFNs and immune-inhibitory receptors in melanoma pathogenesis, which serve as targets for combination immunotherapies. Using a genetically engineered mouse melanoma model, we demonstrate that targeted activation of the type I IFN system with immunostimulatory RNA in combination with blockade of immune-inhibitory receptors is a rational strategy to expose immune cell-poor tumors to cellular immune surveillance. ©2014 American Association for Cancer Research.

  14. Transmigration characteristics of breast cancer and melanoma cells through the brain endothelium: Role of Rac and PI3K.

    PubMed

    Molnár, Judit; Fazakas, Csilla; Haskó, János; Sipos, Orsolya; Nagy, Krisztina; Nyúl-Tóth, Ádám; Farkas, Attila E; Végh, Attila G; Váró, György; Galajda, Péter; Krizbai, István A; Wilhelm, Imola

    2016-05-03

    Brain metastases are common and devastating complications of both breast cancer and melanoma. Although mammary carcinoma brain metastases are more frequent than those originating from melanoma, this latter has the highest tropism to the brain. Using static and dynamic in vitro approaches, here we show that melanoma cells have increased adhesion to the brain endothelium in comparison to breast cancer cells. Moreover, melanoma cells can transmigrate more rapidly and in a higher number through brain endothelial monolayers than breast cancer cells. In addition, melanoma cells have increased ability to impair tight junctions of cerebral endothelial cells. We also show that inhibition of Rac or PI3K impedes adhesion of breast cancer cells and melanoma cells to the brain endothelium. In addition, inhibition of Rac or PI3K inhibits the late phase of transmigration of breast cancer cells and the early phase of transmigration of melanoma cells. On the other hand, the Rac inhibitor EHT1864 impairs the junctional integrity of the brain endothelium, while the PI3K inhibitor LY294002 has no damaging effect on interendothelial junctions. We suggest that targeting the PI3K/Akt pathway may represent a novel opportunity in preventing the formation of brain metastases of melanoma and breast cancer.

  15. SIRT1 deacetylase is overexpressed in human melanoma and its small molecule inhibition imparts anti-proliferative response via p53 activation.

    PubMed

    Wilking, Melissa J; Singh, Chandra; Nihal, Minakshi; Zhong, Weixiong; Ahmad, Nihal

    2014-12-01

    Melanoma causes more deaths than any other skin cancer, and its incidence in the US continues to rise. Current medical therapies are insufficient to control this deadly neoplasm, necessitating the development of new target-based approaches. The objective of this study was to determine the role and functional significance of the class III histone deacetylase SIRT1 in melanoma. We have found that SIRT1 is overexpressed in clinical human melanoma tissues and human melanoma cell lines (Sk-Mel-2, WM35, G361, A375, and Hs294T) compared to normal skin and normal melanocytes, respectively. In addition, treatment of melanoma cell lines A375, Hs294T, and G361 with Tenovin-1, a small molecule SIRT1 inhibitor, resulted in a significant decrease in cell growth and cell viability. Further, Tenovin-1 treatment also resulted in a marked decrease in the clonogenic survival of melanoma cells. Further experiments showed that the anti-proliferative response of Tenovin-1 was accompanied by an increase in the protein as well as activity of the tumor suppressor p53. This increase in p53 activity was substantiated by an increase in the protein level of its downstream target p21. Overall, these data suggest that small molecule inhibition of SIRT1 causes anti-proliferative effects in melanoma cells. SIRT1 appears to be acting through the activity of the tumor suppressor p53, which is not mutated in the majority of melanomas. However, future detailed studies are needed to further explore the role and mechanism of SIRT1 in melanoma development and progression and its usefulness in melanoma treatment.

  16. Convection-enhanced delivery of sorafenib and suppression of tumor progression in a murine model of brain melanoma through the inhibition of signal transducer and activator of transcription 3.

    PubMed

    Zou, Zhaoxia; Yin, Yufang; Lin, Jenny; Hsu, Li-Chen J; Brandon, Vanessa L; Yang, Fan; Jove, Richard; Jandial, Rahul; Li, Gang; Chen, Mike Y

    2016-05-01

    OBJECT Despite recent advances, metastatic melanoma remains a terminal disease, in which life-threatening brain metastasis occurs in approximately half of patients. Sorafenib is a multikinase inhibitor that induces apoptosis of melanoma cells in vitro. However, systemic administration has been ineffective because adequate tissue concentrations cannot be achieved. This study investigated if convection-enhanced delivery (CED) of sorafenib would enhance tumor control and survival via inhibition of the signal transducer and activator of transcription 3 (Stat3) pathway in a murine model of metastatic brain melanoma. METHODS Melanoma cells treated with sorafenib in vitro were examined for signaling and survival changes. The effect of sorafenib given by CED was assessed by bioluminescent imaging and animal survival. RESULTS The results showed that sorafenib induced cell death in the 4 established melanoma cell lines and in 1 primary cultured melanoma cell line. Sorafenib inhibited Stat3 phosphorylation in HTB65, WYC1, and B16 cells. Accordingly, sorafenib treatment also decreased expression of Mcl-1 mRNA in melanoma cell lines. Because sorafenib targets multiple pathways, the present study demonstrated the contribution of the Stat3 pathway by showing that mouse embryonic fibroblast (MEF) Stat3 +/+ cells were significantly more sensitive to sorafenib than MEF Stat3 -/- cells. In the murine model of melanoma brain metastasis used in this study, CED of sorafenib increased survival by 150% in the treatment group compared with animals receiving the vehicle control (p < 0.01). CED of sorafenib also significantly abrogated tumor growth. CONCLUSIONS The data from this study indicate that local delivery of sorafenib effectively controls brain melanoma. These findings validate further investigation of the use of CED to distribute molecularly targeted agents.

  17. Ginsenoside G-Rh2 synergizes with SMI-4a in anti-melanoma activity through autophagic cell death.

    PubMed

    Lv, Da-Lun; Chen, Lei; Ding, Wei; Zhang, Wei; Wang, He-Li; Wang, Shuai; Liu, Wen-Bei

    2018-01-01

    Melanoma is a leading cause of cancer death worldwide, and SMI-4a and G-Rh2 exert anti-tumor activity in multiple cancer. However, SMI-4a as well as a synergistic relationship between SMI-4a and G-Rh2 in anti-melanoma capacity are still unknown. Therefore, we investigated the effects of SMI-4a and combined SMI-4a with G-Rh2 on the viability, apoptosis and autophagy of melanoma, and to preliminarily explore the underlying mechanism of SMI-4a and combined SMI-4a with G-Rh2 in inhibiting tumor growth. Cell viability was examined with cell counting Kit 8 assay and colony formation assay; Apoptosis was evaluated by flow cytometry and Caspase 3/7 activity assay; Western blotting was used to test proteins related to autophagy and the AKT/mammalian target of rapamycin (mTOR) signaling pathway; Tumor xenograft model in BALB/c nude mice was performed to evaluate the effects of SMI-4a and combined SMI-4a with G-Rh2 in anti-melanoma in vivo. SMI-4a, a pharmacological inhibitor of PIM-1, could decrease cell viability, induce apoptosis, and promote Caspase 3/7 activity in both A375 and G361 melanoma cells, and SMI-4a inhibited tumor growth by inducing autophagy via down-regulating AKT/mTOR axis in melanoma cells. Furthermore, G-Rh2 amplified the anti-tumor activity of SMI-4a in melanoma cells via strengthening autophagy. Our results suggested that SMI-4a could enhance autophagy-inducing apoptosis by inhibiting AKT/mTOR signaling pathway in melanoma cells, and G-Rh2 could enhance the effects of SMI-4a against melanoma cancer via amplifying autophagy induction. This study demonstrates that combined SMI-4a and G-Rh2 might be a novel alternative strategy for melanoma treatment.

  18. De-adhesion dynamics of melanoma cells from brain endothelial layer.

    PubMed

    Varga, Béla; Domokos, Réka Anita; Fazakas, Csilla; Wilhelm, Imola; Krizbai, István A; Szegletes, Zsolt; Gergely, Csilla; Váró, György; Végh, Attila G

    2018-03-01

    Metastasis formation is a complex and not entirely understood process. The poorest prognosis and the most feared complications are associated to brain metastases. Melanoma derived brain metastases show the highest prevalence. Due to the lack of classical lymphatic drainage, in the process of brain metastases formation the haematogenous route is of primordial importance. The first and crucial step in this multistep process is the establishment of firm adhesion between the blood travelling melanoma cells and the tightly connected layer of the endothelium, which is the fundamental structure of the blood-brain barrier. This study compares the de-adhesion properties and dynamics of three melanoma cells types (WM35, A2058 and A375) to a confluent layer of brain micro-capillary endothelial cells. Cell type dependent adhesion characteristics are presented, pointing towards the existence of metastatic potential related nanomechanical aspects. Apparent mechanical properties such as elasticity, maximal adhesion force, number, size and distance of individual rupture events showed altered values pointing towards cell type dependent aspects. Our results underline the importance of mechanical details in case of intercellular interactions. Nevertheless, it suggests that in adequate circumstances elastic and adhesive characterizations might be used as biomarkers. Copyright © 2017. Published by Elsevier B.V.

  19. Two novel atypical PKC inhibitors; ACPD and DNDA effectively mitigate cell proliferation and epithelial to mesenchymal transition of metastatic melanoma while inducing apoptosis

    PubMed Central

    Ratnayake, Wishrawana S.; Apostolatos, André H.; Ostrov, David A.; Acevedo-Duncan, Mildred

    2017-01-01

    Atypical protein kinase Cs (aPKC) are involved in cell cycle progression, tumorigenesis, cell survival and migration in many cancers. We believe that aPKCs play an important role in cell motility of melanoma by regulating cell signaling pathways and inducing epithelial to mesenchymal transition (EMT). We have investigated the effects of two novel aPKC inhibitors; 2-acetyl-1,3-cyclopentanedione (ACPD) and 3,4-diaminonaphthalene-2,7-disulfonic acid (DNDA) on cell proliferation, apoptosis, migration and invasion of two malignant melanoma cell lines compared to normal melanocytes. Molecular docking data suggested that both inhibitors specifically bind to protein kinase C-zeta (PKC-ζ) and PKC-iota (PKC-ι) and kinase activity assays were carried out to confirm these observations. Both inhibitors decreased the levels of total and phosphorylated PKC-ζ and PKC-ι. Increased levels of E-cadherin, RhoA, PTEN and decreased levels of phosphorylated vimentin, total vimentin, CD44, β-catenin and phosphorylated AKT in inhibitor treated cells. This suggests that inhibition of both PKC-ζ and PKC-ι using ACPD and DNDA downregulates EMT and induces apoptosis in melanoma cells. We also carried out PKC-ι and PKC-ζ directed siRNA treatments to prove the above observations. Immunoprecipitation data suggested an association between PKC-ι and vimentin and PKC-ι siRNA treatments confirmed that PKC-ι activates vimentin by phosphorylation. These results further suggested that PKC-ι is involved in signaling pathways which upregulate EMT and which can be effectively suppressed using ACPD and DNDA. Our results summarize that melanoma cells proliferate via aPKC/AKT/NF-κB mediated pathway while inducing the EMT via PKC-ι/Par6/RhoA pathway. Overall, results show that aPKCs are essential for melanoma progression and metastasis, suggesting that ACPD and DNDA can be effectively used as potential therapeutic drugs for melanoma by inhibiting aPKCs. PMID:29048609

  20. Expression of monoacylglycerol lipase as a marker of tumour invasion and progression in malignant melanoma.

    PubMed

    Baba, Yuko; Funakoshi, T; Mori, M; Emoto, K; Masugi, Y; Ekmekcioglu, S; Amagai, M; Tanese, K

    2017-12-01

    Accumulating evidence suggests that the lipid lytic enzyme monoacylglycerol lipase (MAGL) promotes tumour invasion and metastasis through up-regulation of pro-tumorigenic signalling lipids in several tumour cell lines. However, the expression status of MAGL in clinical melanoma tissues and its clinicopathological significance remain unclear. To correlate the tumour expression status of MAGL with the clinicopathological information of patients with malignant melanoma. Polymerase chain reaction (PCR) array screening was performed, and the results were validated using immunocytochemical analysis of tumour and non-tumour melanocytic cell lines. Immunohistochemical staining for MAGL was performed for 74 melanoma samples, including 48 primary and 26 metastatic tumours, in which the expression of MAGL was determined by evaluating the percentage of MAGL-positive tumour cells and the MAGL staining intensity. Finally, we analysed the association of MAGL expression status with tumour progression, tumour thickness and vascular invasion of the primary lesion. Immunocytochemical analysis revealed that MAGL was expressed in all 12 melanoma cell lines, but not in normal human epidermal melanocytes. In the immunohistochemical analysis, positive staining for MAGL was noted in 32 of 48 (64.5%) primary lesions, 14 of 17 (82.4%) lymph node metastatic lesions and 7 of 9 (77.8%) skin metastatic lesions. Metastatic tumours had a significantly higher staining intensity (P = 0.033 for lymph node, P = 0.010 for skin). In the analysis of primary lesions, higher MAGL expression correlated with greater tumour thickness (P = 0.015) and the presence of vascular invasion (P = 0.017). On further evaluation of MAGL-positive primary lesions, staining intensity of MAGL tended to be higher in deeper areas of the tumour mass. The expression of MAGL in tumour cells reflects the aggressiveness of melanoma cells and may serve as a marker of tumour progression. © 2017 European Academy of Dermatology and

  1. Resveratrol inhibits uveal melanoma tumor growth via early mitochondrial dysfunction.

    PubMed

    van Ginkel, Paul R; Darjatmoko, Soesiawati R; Sareen, Dhruv; Subramanian, Lalita; Bhattacharya, Saswati; Lindstrom, Mary J; Albert, Daniel M; Polans, Arthur S

    2008-04-01

    To test the efficacy of resveratrol, a nontoxic plant product, in the treatment of uveal melanoma. The effect of oral administration and peritumor injection of resveratrol was tested on tumor growth in two animal models of uveal melanoma. The mechanism of resveratrol action on uveal melanoma cells was studied in vitro in a cell-viability assay: with JC-1 dye, to measure mitochondrial membrane potential; by Western blot analysis, to analyze the cellular redistribution of cytochrome c and Smac/diablo; and in a fluorescence assay with specific substrates, to measure activation of different caspases. Resveratrol treatment inhibited tumor growth in animal models of uveal melanoma. Since oral administration resulted in relatively low bioavailability of resveratrol, the effect of increased local levels was tested by peritumor injection of the drug. This method resulted in tumor cell death and tumor regression. In vitro experiments with multiple uveal melanoma cell lines demonstrate that resveratrol causes a decrease in cell viability, resulting at least in part from an increase in apoptosis through a mitochondrial pathway. An early event in drug action is the direct targeting of mitochondria by resveratrol, which leads to a decrease in mitochondrial membrane potential and the eventual activation of caspase-3. These data suggest that resveratrol can inhibit tumor growth and can induce apoptosis via the intrinsic mitochondrial pathway and that by further increasing bioavailability of resveratrol the potency of the drug can be increased, leading to tumor regression. The nontoxic nature of the drug at levels needed for therapy make resveratrol an attractive candidate for the treatment of uveal melanoma.

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

    PubMed

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

    1998-03-01

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

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

    PubMed

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

    2017-01-01

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

  4. Melanoma-specific MHC-II expression represents a tumour-autonomous phenotype and predicts response to anti-PD-1/PD-L1 therapy

    PubMed Central

    Johnson, Douglas B.; Estrada, Monica V.; Salgado, Roberto; Sanchez, Violeta; Doxie, Deon B.; Opalenik, Susan R.; Vilgelm, Anna E.; Feld, Emily; Johnson, Adam S.; Greenplate, Allison R.; Sanders, Melinda E.; Lovly, Christine M.; Frederick, Dennie T.; Kelley, Mark C.; Richmond, Ann; Irish, Jonathan M.; Shyr, Yu; Sullivan, Ryan J.; Puzanov, Igor; Sosman, Jeffrey A.; Balko, Justin M.

    2016-01-01

    Anti-PD-1 therapy yields objective clinical responses in 30–40% of advanced melanoma patients. Since most patients do not respond, predictive biomarkers to guide treatment selection are needed. We hypothesize that MHC-I/II expression is required for tumour antigen presentation and may predict anti-PD-1 therapy response. In this study, across 60 melanoma cell lines, we find bimodal expression patterns of MHC-II, while MHC-I expression was ubiquitous. A unique subset of melanomas are capable of expressing MHC-II under basal or IFNγ-stimulated conditions. Using pathway analysis, we show that MHC-II(+) cell lines demonstrate signatures of ‘PD-1 signalling', ‘allograft rejection' and ‘T-cell receptor signalling', among others. In two independent cohorts of anti-PD-1-treated melanoma patients, MHC-II positivity on tumour cells is associated with therapeutic response, progression-free and overall survival, as well as CD4+ and CD8+ tumour infiltrate. MHC-II+ tumours can be identified by melanoma-specific immunohistochemistry using commercially available antibodies for HLA-DR to improve anti-PD-1 patient selection. PMID:26822383

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

    PubMed

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

    2010-10-01

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

  6. At the bench: adoptive cell therapy for melanoma.

    PubMed

    Urba, Walter J

    2014-06-01

    The cellular and molecular principles that furnish the foundation for ACT of melanoma and their implications for further clinical research are reviewed. The parallel advances in basic immunology, preclinical animal studies, and clinical trials over the last two decades have been integrated successfully with improvements in technology to produce an effective ACT strategy for patients with melanoma. From the initial observation that tumors could be treated effectively by the transfer of immune cells to current strategies using preconditioning with myeloablative therapy before adoptive transfer of native or genetically altered T cells, the role of preclinical animal models is discussed. The importance of the pmel transgenic mouse model in the determination of the mechanisms of lymphodepletion, the ongoing work to identify the optimal T cells for adoptive immunotherapy, and the early impact of the emerging discipline of synthetic biology are highlighted. The clinical consequences of the research described herein are reviewed in the companion manuscript. © 2014 Society for Leukocyte Biology.

  7. MST1 activation by curcumin mediates JNK activation, Foxo3a nuclear translocation and apoptosis in melanoma cells

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

    Yu, Teng, E-mail: tengyu33@yahoo.com; Ji, Jiang; Guo, Yong-li

    2013-11-08

    Highlights: •Curcumin activates MST1 in melanoma cells. •MST1 mediates curcumin-induced apoptosis of melanoma cells. •ROS production is involved in curcumin-induced MST1 activation. •MST1 mediates curcumin-induced JNK activation in melanoma cells. •MST1 mediates curcumin-induced Foxo3a nuclear translocation and Bim expression. -- Abstract: Different groups including ours have shown that curcumin induces melanoma cell apoptosis, here we focused the role of mammalian Sterile 20-like kinase 1 (MST1) in it. We observed that curcumin activated MST1-dependent apoptosis in cultured melanoma cells. MST1 silencing by RNA interference (RNAi) suppressed curcumin-induced cell apoptosis, while MST1 over-expressing increased curcumin sensitivity. Meanwhile, curcumin induced reactive oxygen speciesmore » (ROS) production in melanoma cells, and the ROS scavenger, N-acetyl-cysteine (NAC), almost blocked MST1 activation to suggest that ROS might be required for MST1 activation by curcumin. c-Jun N-terminal protein kinase (JNK) activation by curcumin was dependent on MST1, since MST1 inhibition by RNAi or NAC largely inhibited curcumin-induced JNK activation. Further, curcumin induced Foxo3 nuclear translocation and Bim-1 (Foxo3 target gene) expression in melanoma cells, such an effect by curcumin was inhibited by MST1 RNAi. In conclusion, we suggested that MST1 activation by curcumin mediates JNK activation, Foxo3a nuclear translocation and apoptosis in melanoma cells.« less

  8. Differential effects of selenite and selenate on human melanocytes, keratinocytes, and melanoma cells.

    PubMed

    Bandura, Laura; Drukala, Justyna; Wolnicka-Glubisz, Agnieszka; Björnstedt, Mikael; Korohoda, Wlodzimierz

    2005-04-01

    Among the substances that attracted the attention of oncologists in recent years are selenium-containing compounds, both inorganic and organic. Several epidemiological studies have shown an inverse correlation between selenium intake and cancer incidence. In the experiments reported here, we compared the effects of 2 inorganic selenium-containing salts that differed in the level of selenium oxidation, selenite IV and selenate VI. We tested the effects of these 2 compounds on cell survival and growth, cell cycle processing, cell morphology, cytoskeleton, and lipid peroxidation in 3 human skin cell types: normal keratinocytes, melanocytes, and human melanoma cell line HTB140. The different effects of selenite and selenate on the viability, growth, and morphology of normal cells and tumor cells are reported and provide a base for future research and treatment of some neoplastic diseases. The attention is paid to cell apoptosis induced by selenite and not by selenate, and the effects of tested substances on thioredoxin reductase system are postulated.

  9. Modelling cell population growth with applications to cancer therapy in human tumour cell lines.

    PubMed

    Basse, Britta; Baguley, Bruce C; Marshall, Elaine S; Wake, Graeme C; Wall, David J N

    2004-01-01

    In this paper we present an overview of the work undertaken to model a population of cells and the effects of cancer therapy. We began with a theoretical one compartment size structured cell population model and investigated its asymptotic steady size distributions (SSDs) (On a cell growth model for plankton, MMB JIMA 21 (2004) 49). However these size distributions are not similar to the DNA (size) distributions obtained experimentally via the flow cytometric analysis of human tumour cell lines (data obtained from the Auckland Cancer Society Research Centre, New Zealand). In our one compartment model, size was a generic term, but in order to obtain realistic steady size distributions we chose size to be DNA content and devised a multi-compartment mathematical model for the cell division cycle where each compartment corresponds to a distinct phase of the cell cycle (J. Math. Biol. 47 (2003) 295). We then incorporated another compartment describing the possible induction of apoptosis (cell death) from mitosis phase (Modelling cell death in human tumour cell lines exposed to anticancer drug paclitaxel, J. Math. Biol. 2004, in press). This enabled us to compare our model to flow cytometric data of a melanoma cell line where the anticancer drug, paclitaxel, had been added. The model gives a dynamic picture of the effects of paclitaxel on the cell cycle. We hope to use the model to describe the effects of other cancer therapies on a number of different cell lines. Copyright 2004 Elsevier Ltd.

  10. Phenotype and function of T cells infiltrating visceral metastases from gastrointestinal cancers and melanoma: implications for adoptive cell transfer therapy.

    PubMed

    Turcotte, Simon; Gros, Alena; Hogan, Katherine; Tran, Eric; Hinrichs, Christian S; Wunderlich, John R; Dudley, Mark E; Rosenberg, Steven A

    2013-09-01

    Adoptive cell transfer of tumor-infiltrating lymphocytes (TILs) can mediate cancer regression in patients with metastatic melanoma, but whether this approach can be applied to common epithelial malignancies remains unclear. In this study, we compared the phenotype and function of TILs derived from liver and lung metastases from patients with gastrointestinal (GI) cancers (n = 14) or melanoma (n = 42). Fewer CD3(+) T cells were found to infiltrate GI compared with melanoma metastases, but the proportions of CD8(+) cells, T cell differentiation stage, and expression of costimulatory molecules were similar for both tumor types. Clinical-scale expansion up to ~50 × 10(9) T cells on average was obtained for all patients with GI cancer and melanoma. From GI tumors, however, TIL outgrowth in high-dose IL-2 yielded 22 ± 1.4% CD3(+)CD8(+) cells compared with 63 ± 2.4% from melanoma (p < 0.001). IFN-γ ELISA demonstrated MHC class I-mediated reactivity of TIL against autologous tumor in 5 of 7 GI cancer patients tested (9% of 188 distinct TIL cultures) and in 9 of 10 melanoma patients (43% of 246 distinct TIL cultures). In these assays, MHC class I-mediated up-regulation of CD137 (4-1BB) expression on CD8(+) cells suggested that 0-3% of TILs expanded from GI cancer metastases were tumor-reactive. This study implies that the main challenge to the development of TIL adoptive cell transfer for metastatic GI cancers may not be the in vitro expansion of bulk TILs, but the ability to select and enrich for tumor-reactive T cells.

  11. Phenotype and Function of T Cells Infiltrating Visceral Metastases from Gastrointestinal Cancers and Melanoma: Implications for Adoptive Cell Transfer Therapy

    PubMed Central

    Turcotte, Simon; Gros, Alena; Hogan, Katherine; Tran, Eric; Hinrichs, Christian S.; Wunderlich, John R.; Dudley, Mark E.

    2013-01-01

    Adoptive cell transfer of tumor-infiltrating lymphocytes (TILs) can mediate cancer regression in patients with metastatic melanoma, but whether this approach can be applied to common epithelial malignancies remains unclear. In this study, we compared the phenotype and function of TILs derived from liver and lung metastases from patients with gastrointestinal (GI) cancers (n = 14) or melanoma (n = 42). Fewer CD3+ T cells were found to infiltrate GI compared with melanoma metastases, but the proportions of CD8+ cells, T cell differentiation stage, and expression of costimulatory molecules were similar for both tumor types. Clinical-scale expansion up to ∼50 × 109 T cells on average was obtained for all patients with GI cancer and melanoma. From GI tumors, however, TIL outgrowth in high-dose IL-2 yielded 22 ± 1.4% CD3+CD8+ cells compared with 63 ± 2.4% from melanoma (p < 0.001). IFN-γ ELISA demonstrated MHC class I–mediated reactivity of TIL against autologous tumor in 5 of 7 GI cancer patients tested (9% of 188 distinct TIL cultures) and in 9 of 10 melanoma patients (43% of 246 distinct TIL cultures). In these assays, MHC class I–mediated up-regulation of CD137 (4-1BB) expression on CD8+ cells suggested that 0–3% of TILs expanded from GI cancer metastases were tumor-reactive. This study implies that the main challenge to the development of TIL adoptive cell transfer for metastatic GI cancers may not be the in vitro expansion of bulk TILs, but the ability to select and enrich for tumor-reactive T cells. PMID:23904171

  12. Protective CD8 Memory T Cell Responses to Mouse Melanoma Are Generated in the Absence of CD4 T Cell Help

    PubMed Central

    Steinberg, Shannon M.; Zhang, Peisheng; Turk, Mary Jo

    2011-01-01

    Background We have previously demonstrated that temporary depletion of CD4 T cells in mice with progressive B16 melanoma, followed by surgical tumor excision, induces protective memory CD8 T cell responses to melanoma/melanocyte antigens. We also showed that persistence of these CD8 T cells is supported, in an antigen-dependent fashion, by concurrent autoimmune melanocyte destruction. Herein we explore the requirement of CD4 T cell help in priming and maintaining this protective CD8 T cell response to melanoma. Methodology and Principal Findings To induce melanoma/melanocyte antigen-specific CD8 T cells, B16 tumor bearing mice were depleted of regulatory T cells (Treg) by either temporary, or long-term continuous treatment with anti-CD4 (mAb clone GK1.5). Total depletion of CD4 T cells led to significant priming of IFN-γ-producing CD8 T cell responses to TRP-2 and gp100. Surprisingly, treatment with anti-CD25 (mAb clone PC61), to specifically deplete Treg cells while leaving help intact, was ineffective at priming CD8 T cells. Thirty to sixty days after primary tumors were surgically excised, mice completely lacking CD4 T cell help developed autoimmune vitiligo, and maintained antigen-specific memory CD8 T cell responses that were highly effective at producing cytokines (IFN-γ, TNF-α, and IL-2). Mice lacking total CD4 T cell help also mounted protection against re-challenge with B16 melanoma sixty days after primary tumor excision. Conclusions and Significance This work establishes that CD4 T cell help is dispensable for the generation of protective memory T cell responses to melanoma. Our findings support further use of CD4 T cell depletion therapy for inducing long-lived immunity to cancer. PMID:22046294

  13. Intratumoral genetic heterogeneity in metastatic melanoma is accompanied by variation in malignant behaviors.

    PubMed

    Anaka, Matthew; Hudson, Christopher; Lo, Pu-Han; Do, Hongdo; Caballero, Otavia L; Davis, Ian D; Dobrovic, Alexander; Cebon, Jonathan; Behren, Andreas

    2013-10-11

    Intratumoral heterogeneity is a major obstacle for the treatment of cancer, as the presence of even minor populations that are insensitive to therapy can lead to disease relapse. Increased clonal diversity has been correlated with a poor prognosis for cancer patients, and we therefore examined genetic, transcriptional, and functional diversity in metastatic melanoma. Amplicon sequencing and SNP microarrays were used to profile somatic mutations and DNA copy number changes in multiple regions from metastatic lesions. Clonal genetic and transcriptional heterogeneity was also assessed in single cell clones from early passage cell lines, which were then subjected to clonogenicity and drug sensitivity assays. MAPK pathway and tumor suppressor mutations were identified in all regions of the melanoma metastases analyzed. In contrast, we identified copy number abnormalities present in only some regions in addition to homogeneously present changes, suggesting ongoing genetic evolution following metastatic spread. Copy number heterogeneity from a tumor was represented in matched cell line clones, which also varied in their clonogenicity and drug sensitivity. Minor clones were identified based on dissimilarity to the parental cell line, and these clones were the most clonogenic and least sensitive to drugs. Finally, treatment of a polyclonal cell line with paclitaxel to enrich for drug-resistant cells resulted in the adoption of a gene expression profile with features of one of the minor clones, supporting the idea that these populations can mediate disease relapse. Our results support the hypothesis that minor clones might have major consequences for patient outcomes in melanoma.

  14. Intratumoral genetic heterogeneity in metastatic melanoma is accompanied by variation in malignant behaviors

    PubMed Central

    2013-01-01

    Background Intratumoral heterogeneity is a major obstacle for the treatment of cancer, as the presence of even minor populations that are insensitive to therapy can lead to disease relapse. Increased clonal diversity has been correlated with a poor prognosis for cancer patients, and we therefore examined genetic, transcriptional, and functional diversity in metastatic melanoma. Methods Amplicon sequencing and SNP microarrays were used to profile somatic mutations and DNA copy number changes in multiple regions from metastatic lesions. Clonal genetic and transcriptional heterogeneity was also assessed in single cell clones from early passage cell lines, which were then subjected to clonogenicity and drug sensitivity assays. Results MAPK pathway and tumor suppressor mutations were identified in all regions of the melanoma metastases analyzed. In contrast, we identified copy number abnormalities present in only some regions in addition to homogeneously present changes, suggesting ongoing genetic evolution following metastatic spread. Copy number heterogeneity from a tumor was represented in matched cell line clones, which also varied in their clonogenicity and drug sensitivity. Minor clones were identified based on dissimilarity to the parental cell line, and these clones were the most clonogenic and least sensitive to drugs. Finally, treatment of a polyclonal cell line with paclitaxel to enrich for drug-resistant cells resulted in the adoption of a gene expression profile with features of one of the minor clones, supporting the idea that these populations can mediate disease relapse. Conclusion Our results support the hypothesis that minor clones might have major consequences for patient outcomes in melanoma. PMID:24119551

  15. MicroRNA-365 inhibits growth, invasion and metastasis of malignant melanoma by targeting NRP1 expression.

    PubMed

    Bai, Juanjuan; Zhang, Zhongling; Li, Xing; Liu, Huifan

    2015-01-01

    The role of miR-365 in cancer cells seemed controversial in previous studies. We thereby in this article aimed to define the role of miR-365 in malignant melanoma (MM) pathogenesis. We detected miR-365 expression in malignant melanoma cell lines and then investigated the effects of miR-365 on the metastasis and malignancy of melanoma cells. The correlation between miR-365 level and NRP1 (neuropilin1) was further investigated in clinical malignant melanoma specimens. MiR-365 was strongly down-regulated in malignant melanoma (MM) tissues and cell lines, and its expression levels were associated with lymph node metastasis and clinical stage, as well as overall survival and replase-free survival of MM. We also found that ectopic expression of miR-365 inhibited MM cell proliferation and MM metastasis in vitro and in vivo. We further identified a novel mechanism of miR-365 to suppress MM growth and metastasis. NRP1 was proved to be a direct target of miR-365, using luciferase assay and western blot. NRP1 over-expression in miR-365 expressing cells could rescue invasion and growth defects of miR-365. In addition, miR-365 expression inversely correlated with NRP1 protein levels in MM. Our data suggest that miR-365 functions as a tumor suppressor in MM development and progression, and holds promise as a prognostic biomarker and potential therapeutic target for MM.

  16. Phenformin enhances the therapeutic benefit of BRAF(V600E) inhibition in melanoma.

    PubMed

    Yuan, Ping; Ito, Koichi; Perez-Lorenzo, Rolando; Del Guzzo, Christina; Lee, Jung Hyun; Shen, Che-Hung; Bosenberg, Marcus W; McMahon, Martin; Cantley, Lewis C; Zheng, Bin

    2013-11-05

    Biguanides, such as the diabetes therapeutics metformin and phenformin, have demonstrated antitumor activity both in vitro and in vivo. The energy-sensing AMP-activated protein kinase (AMPK) is known to be a major cellular target of biguanides. Based on our discovery of cross-talk between the AMPK and v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) signaling pathways, we investigated the antitumor effects of combining phenformin with a BRAF inhibitor PLX4720 on the proliferation of BRAF-mutated melanoma cells in vitro and on BRAF-driven tumor growth in vivo. Cotreatment of BRAF-mutated melanoma cell lines with phenformin and PLX4720 resulted in synergistic inhibition of cell viability, compared with the effects of the single agent alone. Moreover, treatment with phenformin significantly delayed the development of resistance to PLX4720 in cultured melanoma cells. Biochemical analyses showed that phenformin and PLX4720 exerted cooperative effects on inhibiting mTOR signaling and inducing apoptosis. Noticeably, phenformin selectively targeted subpopulations of cells expressing JARID1B, a marker for slow cycling melanoma cells, whereas PLX4720 selectively targeted JARID1B-negative cells. Finally, in contrast to their use as single agents, the combination of phenformin and PLX4720 induced tumor regression in both nude mice bearing melanoma xenografts and in a genetically engineered BRAF(V600E)/PTEN(null)-driven mouse model of melanoma. These results strongly suggest that significant therapeutic advantage may be achieved by combining AMPK activators such as phenformin with BRAF inhbitors for the treatment of melanoma.

  17. Host-Derived Pericytes and Sca-1+ Cells Predominate in the MART-1− Stroma Fraction of Experimentally Induced Melanoma

    PubMed Central

    Treviño-Villarreal, J. Humberto; Cotanche, Douglas A.; Sepúlveda, Rosalinda; Bortoni, Magda E.; Manneberg, Otto; Udagawa, Taturo

    2011-01-01

    Identification of cell types in tumor-associated stroma that are involved in the development of melanoma is hampered by their heterogeneity. The authors used flow cytometry and immunohistochemistry to demonstrate that anti–MART-1 antibodies can discriminate between melanoma and stroma cells. They investigated the cellular composition of the MART-1−, non-hematopoietic melanoma-associated stroma, finding it consisted mainly of Sca-1+ and CD146+ cells. These cell types were also observed in the skin and muscle adjacent to developing melanomas. The Sca-1+ cell population was observed distributed in the epidermis, hair follicle bulges, and tumor capsule. The CD146+ population was found distributed within the tumor, mainly associated with blood vessels in a perivascular location. In addition to a perivascular distribution, CD146+ cells expressed α-smooth muscle actin, lacked expression of endothelial markers CD31 and CD34, and were therefore identified as pericytes. Pericytes were found to be associated with CD31+ endothelial cells; however, some pericytes were also observed associated with CD31−, MART-1+ B16 melanoma cells that appeared to form blood vessel structures. Furthermore, the authors observed extensive nuclear expression of HIF-1α in melanoma and stroma cells, suggesting hypoxia is an important factor associated with the melanoma microenvironment and vascularization. The results suggest that pericytes and Sca-1+ stroma cells are important contributors to melanoma development. PMID:22147606

  18. Oral picropodophyllin (PPP) is well tolerated in vivo and inhibits IGF-1R expression and growth of uveal melanoma.

    PubMed

    Economou, Mario A; Andersson, Sandra; Vasilcanu, Diana; All-Ericsson, Charlotta; Menu, Eline; Girnita, Ada; Girnita, Leonard; Axelson, Magnus; Seregard, Stefan; Larsson, Olle

    2008-06-01

    The cyclolignan picropodophyllin (PPP) efficiently blocks the activity of insulinlike growth factor-1 receptor (IGF-1R) and inhibits the growth of uveal melanoma cells in vitro and in vivo. In this study, the authors investigated the efficiency of orally administered PPP on the growth of uveal melanoma xenografts. In addition, they focused on the effect of PPP on vascular endothelial growth factor (VEGF) in vivo and evaluated its effects in combination with other established antitumor agents in vitro. Four different uveal melanoma cell lines (OCM-1, OCM-3, OCM-8, 92-1) were treated with PPP alone and in combination with imatinib mesylate, cisplatin, 5-fluorouracil, and doxorubicin. Cell viability was determined by XTT assay. SCID mice that underwent xenografting with uveal melanoma cells were used to determine antitumor efficacy of oral PPP in vivo. Five mice were used per group. Tumor samples obtained from the in vivo experiments were analyzed for VEGF and IGF-1R expression by Western blotting. PPP was found to be superior to the other antitumor agents in killing uveal melanoma cells in all four cell lines (IC50 < 0.05 microM). Oral PPP inhibited uveal melanoma growth in vivo in OCM-3 (P = 0.03) and OCM-8 (P = 0.01) xenografts and was well tolerated by the animals. PPP decreased VEGF expression in the OCM-1 (P = 0.006) and OCM-8 (P = 0.01) tumors. Oral PPP was well tolerated in vivo, caused total growth inhibition of uveal melanoma xenografts, and decreased VEGF levels in the tumors.

  19. Metastatic melanoma cells with BRAF G469A mutation: nab-paclitaxel better than vemurafenib?

    PubMed

    Porcelli, Letizia; Guida, Gabriella; Tommasi, Stefania; Guida, Michele; Azzariti, Amalia

    2015-08-01

    BRAF G469A is a missense mutation within exon 11 of the BRAF gene resulting in a constitutively activated enzyme frequently associated with MAP kinase cascade signaling activation. No evidence currently exists about its role in determining sensitivity/resistance to BRAF inhibitors, utilized in the treatment of patients carrying BRAF V600 mutations, and to chemotherapy. The newly established metastatic melanoma (MM) cell line MO-1 was characterized for its sensitivity to vemurafenib and nab-paclitaxel, both already utilized for the treatment of MM. All analyses were carried out by comparing results with those found in MM cells wild type for BRAF or mutated in V600. In addition, cellular effectors were investigated by ELISA kits, western blotting and flow cytometry. The exposure to vemurafenib inhibited MO-1 cell proliferation at concentrations similar to those obtained in vemurafenib-resistant melanoma models, and an explanation of this sensitivity is the strong activation of Erk1/2 and the low expression of MITF. Nab-paclitaxel strongly reduced proliferation of MO-1 cells perhaps for the very low expression level of PMEL17, transcriptionally regulated by MITF and negatively involved in determining sensitivity to taxanes. Thus, the mutation BRAF G469A in MM might be related to a weak effectiveness of therapy with BRAF inhibitors and a promising therapeutic approach may be with nab-paclitaxel.

  20. Downregulation of lncRNA H19 inhibits the migration and invasion of melanoma cells by inactivating the NF‑κB and PI3K/Akt signaling pathways.

    PubMed

    Liao, Zhichao; Zhao, Jun; Yang, Yun

    2018-05-01

    As the most aggressive type of skin cancer, melanoma seriously affects human health. Long noncoding (lncRNA) 19 has been demonstrated to be involved in the progression of a number of different types of human cancers. However, the involvement of lncRNA H19 in melanoma remains unknown. Therefore, the present study was performed to investigate the roles of H19 in the development and progression of melanoma. In the present study, 49 patients with melanoma were included. Expression of lncRNA H19 in tumor tissue, adjacent healthy tissue and various cell lines with different treatments was measured by reverse transcription‑quantitative polymerase chain reaction. The effects of H19 knockdown on melanoma cell proliferation, migration and invasion were detected by cell counting kit‑8, wound‑healing and transwell invasion assays, respectively. In addition, the effects of H19 knockdown on the expression of nuclear factor (NF)‑κB pathway‑associated proteins were investigated by western blotting. The results revealed that the expression level of H19 was significantly higher in tumor tissue than in the adjacent healthy tissue of 47 out of 49 patients. H19 knockdown significantly reduced the proliferation, migration and invasion ability of melanoma cells. H19 knockdown also inactivated the phosphoinositide 3‑kinase (PI3K)/protein kinase B (Akt) pathway, which in turn inhibited the activation of the NF‑κB signaling pathway. Thus, downregulation of lncRNA H19 may inhibit the migration and invasion of melanoma cells by inactivating the NF‑κB signaling pathway via the inactivation of the PI3K/Akt signaling pathway. The present study provided references for future studies on the pathogenesis of melanoma and the clinical treatment of this disease.

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

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

    Serafino, A.; Balestrieri, E.; Pierimarchi, P.

    2009-03-10

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

  2. Combinatorial Discovery of Defined Substrates That Promote a Stem Cell State in Malignant Melanoma

    PubMed Central

    2017-01-01

    The tumor microenvironment is implicated in orchestrating cancer cell transformation and metastasis. However, specific cell–ligand interactions between cancer cells and the extracellular matrix are difficult to decipher due to a dynamic and multivariate presentation of many signaling molecules. Here we report a versatile peptide microarray platform that is capable of screening for cancer cell phenotypic changes in response to ligand–receptor interactions. Using a screen of 78 peptide combinations derived from proteins present in the melanoma microenvironment, we identify a proteoglycan binding and bone morphogenic protein 7 (BMP7) derived sequence that selectively promotes the expression of several putative melanoma initiating cell markers. We characterize signaling associated with each of these peptides in the activation of melanoma pro-tumorigenic signaling and reveal a role for proteoglycan mediated adhesion and signaling through Smad 2/3. A defined substratum that controls the state of malignant melanoma may prove useful in spatially normalizing a heterogeneous population of tumor cells for discovery of therapeutics that target a specific state and for identifying new drug targets and reagents for intervention. PMID:28573199

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

    PubMed Central

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

    2012-01-01

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

  4. Genetic Engineering of T Cells to Target HERV-K, an Ancient Retrovirus on Melanoma.

    PubMed

    Krishnamurthy, Janani; Rabinovich, Brian A; Mi, Tiejuan; Switzer, Kirsten C; Olivares, Simon; Maiti, Sourindra N; Plummer, Joshua B; Singh, Harjeet; Kumaresan, Pappanaicken R; Huls, Helen M; Wang-Johanning, Feng; Cooper, Laurence J N

    2015-07-15

    The human endogenous retrovirus (HERV-K) envelope (env) protein is a tumor-associated antigen (TAA) expressed on melanoma but not normal cells. This study was designed to engineer a chimeric antigen receptor (CAR) on T-cell surface, such that they target tumors in advanced stages of melanoma. Expression of HERV-K protein was analyzed in 220 melanoma samples (with various stages of disease) and 139 normal organ donor tissues using immunohistochemical (IHC) analysis. HERV-K env-specific CAR derived from mouse monoclonal antibody was introduced into T cells using the transposon-based Sleeping Beauty (SB) system. HERV-K env-specific CAR(+) T cells were expanded ex vivo on activating and propagating cells (AaPC) and characterized for CAR expression and specificity. This includes evaluating the HERV-K-specific CAR(+) T cells for their ability to kill A375-SM metastasized tumors in a mouse xenograft model. We detected HERV-K env protein on melanoma but not in normal tissues. After electroporation of T cells and selection on HERV-K(+) AaPC, more than 95% of genetically modified T cells expressed the CAR with an effector memory phenotype and lysed HERV-K env(+) tumor targets in an antigen-specific manner. Even though there is apparent shedding of this TAA from tumor cells that can be recognized by HERV-K env-specific CAR(+) T cells, we observed a significant antitumor effect. Adoptive cellular immunotherapy with HERV-K env-specific CAR(+) T cells represents a clinically appealing treatment strategy for advanced-stage melanoma and provides an approach for targeting this TAA on other solid tumors. ©2015 American Association for Cancer Research.

  5. Nucleoli cytomorphology in cutaneous melanoma cells - a new prognostic approach to an old concept.

    PubMed

    Donizy, Piotr; Biecek, Przemyslaw; Halon, Agnieszka; Maciejczyk, Adam; Matkowski, Rafal

    2017-12-29

    The nucleolus is an organelle that is an ultrastructural element of the cell nucleus observed in H&E staining as a roundish body stained with eosin due to its high protein content. Changes in the nucleoli cytomorphology were one of the first histopathological characteristics of malignant tumors. The aim of this study was to assess the relationship between the cytomorphological characteristics of nucleoli and detailed clinicopathological parameters of melanoma patients. Moreover, we analyzed the correlation between cytomorphological parameters of nucleoli and immunoreactivity of selected proteins responsible for, among others, regulation of epithelial-mesenchymal transition (SPARC, N-cadherin), cell adhesion and motility (ALCAM, ADAM-10), mitotic divisions (PLK1), cellular survival (FOXP1) and the functioning of Golgi apparatus (GOLPH3, GP73). Three characteristics of nucleoli - presence, size and number - of cancer cells were assessed in H&E-stained slides of 96 formalin-fixed paraffin-embedded primary cutaneous melanoma tissue specimens. The results were correlated with classical clinicopathological features and patient survival. Immunohistochemical analysis of the above mentioned proteins was described in details in previous studies. Higher prevalence and size of nucleoli were associated with thicker and mitogenic tumors. All three nucleolar characteristics were related to the presence of ulceration. Moreover, microsatellitosis was strongly correlated with the presence of macronucleoli and polynucleolization (presence of two or more nucleoli). Lack of immunologic response manifested as no TILs in primary tumor was associated with high prevalence of melanoma cells with distinct nucleoli. Interestingly, in nodular melanoma a higher percentage of melanoma cells with prominent nucleoli was observed. In Kaplan-Meier analysis, increased prevalence and amount, but not size of nucleoli, were connected with shorter cancer-specific and disease-free survival. (1) High

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

    PubMed

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

    2015-10-27

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

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

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

    Li, Meng; Long, Chaoqin; Yang, Guilan

    2016-03-11

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

  8. Combined activity of temozolomide and the mTOR inhibitor temsirolimus in metastatic melanoma involves DKK1.

    PubMed

    Niessner, Heike; Kosnopfel, Corinna; Sinnberg, Tobias; Beck, Daniela; Krieg, Kathrin; Wanke, Ines; Lasithiotakis, Konstantinos; Bonin, Michael; Garbe, Claus; Meier, Friedegund

    2017-07-01

    The BRAFV600E inhibitor vemurafenib achieves remarkable clinical responses in patients with BRAF-mutant melanoma, but its effects are limited by the onset of drug resistance. In the case of resistance, chemotherapy can still be applied as second line therapy. However, it yields low response rates and strategies are urgently needed to potentiate its effects. In a previous study, we showed that the inhibition of the PI3K-AKT-mTOR pathway significantly increases sensitivity of melanoma cells to chemotherapeutic drugs (J. Invest. Dermatol. 2009, 129, 1500). In this study, the combination of the mTOR inhibitor temsirolimus with the chemotherapeutic agent temozolomide significantly increases growth inhibition and apoptosis in melanoma cells compared to temsirolimus or temozolomide alone. The combination of temozolomide with temsirolimus is not only effective in established but also in newly isolated and vemurafenib-resistant metastatic melanoma cell lines. These effects are associated with the downregulation of the anti-apoptotic protein Mcl-1 and the upregulation of the Wnt antagonist Dickkopf homologue 1 (DKK1). Knock-down of DKK1 suppresses apoptosis induction by the combination of temsirolimus and temozolomide. These data suggest that the inhibition of the mTOR pathway increases sensitivity of melanoma cells towards temozolomide. Chemosensitisation is associated with enhanced expression of the Wnt antagonist DKK1. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  9. Classification of cancer cell lines using matrix-assisted laser desorption/ionization time‑of‑flight mass spectrometry and statistical analysis.

    PubMed

    Serafim, Vlad; Shah, Ajit; Puiu, Maria; Andreescu, Nicoleta; Coricovac, Dorina; Nosyrev, Alexander; Spandidos, Demetrios A; Tsatsakis, Aristides M; Dehelean, Cristina; Pinzaru, Iulia

    2017-10-01

    Over the past decade, matrix-assisted laser desorption/ionization time‑of‑flight mass spectrometry (MALDI‑TOF MS) has been established as a valuable platform for microbial identification, and it is also frequently applied in biology and clinical studies to identify new markers expressed in pathological conditions. The aim of the present study was to assess the potential of using this approach for the classification of cancer cell lines as a quantifiable method for the proteomic profiling of cellular organelles. Intact protein extracts isolated from different tumor cell lines (human and murine) were analyzed using MALDI‑TOF MS and the obtained mass lists were processed using principle component analysis (PCA) within Bruker Biotyper® software. Furthermore, reference spectra were created for each cell line and were used for classification. Based on the intact protein profiles, we were able to differentiate and classify six cancer cell lines: two murine melanoma (B16‑F0 and B164A5), one human melanoma (A375), two human breast carcinoma (MCF7 and MDA‑MB‑231) and one human liver carcinoma (HepG2). The cell lines were classified according to cancer type and the species they originated from, as well as by their metastatic potential, offering the possibility to differentiate non‑invasive from invasive cells. The obtained results pave the way for developing a broad‑based strategy for the identification and classification of cancer cells.

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

    PubMed

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

    2012-03-01

    Melanoma is a lethal form of skin cancer that can metastasize rapidly. While surgery and radiation therapy provide palliative therapy for local tumor growth, systemic therapy is the mainstay of treatment for metastatic melanoma. However, limited chemotherapeutic agents are available for melanoma treatment. In this study, we investigated the anti-melanoma effect of physalin B, the major active compound from a widely used herb medicine, Physalis angulata L. This study demonstrated that physalin B exhibits cytotoxicity towards v-raf murine sarcoma viral oncogene homolog B1 (BRAF)-mutated melanoma A375 and A2058 cells (the IC50 values are lower than 4.6 μg/ml). Cytotoxicity is likely resulted from apoptosis since the apoptotic marker phosphatidylserine are detected immediately under physalin B treatment and apoptotic cells formation. Further examination revealed that physalin B induces expression of the proapoptotic protein NOXA within 2 h and later triggers the expression of Bax and caspase-3 in A375 cells. These results indicate that physalin B can induce apoptosis of melanoma cancer cells via the NOXA, caspase-3, and mitochondria-mediated pathways, but not of human skin fibroblast cells and myoblastic cells. Thus, physalin B has the potential to be developed as an effective chemotherapeutic lead compound for the treatment of malignant melanoma. Copyright © 2011 Elsevier Ltd. All rights reserved.

  11. Label-free longitudinal monitoring of melanogenesis in the evolution of melanoma treatment resistance (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Osseiran, Sam; Wang, Hequn; Dutton-Regester, Ken; Garraway, Levi A.; Evans, Conor L.

    2017-02-01

    While melanoma is not the most common form of skin cancer, it represents the vast majority of skin cancer-related deaths. Indeed, while combination therapies such as Dabrafenib and Trametinib have shown great promise in clinical trials for treating metastatic disease, some melanoma subtypes nevertheless develop resistances to front-line treatments. Under in vitro conditions, some metastatic human melanoma cell lines have been observed to evolve resistance to treatment while simultaneously changing color under brightfield microscopy, hinting at perturbations in pigment synthesis. The process known as melanogenesis gives rise to the two forms of melanin found in mammals: eumelanin, a dark brown/black pigment, and pheomelanin, a much more pale red/blond pigment. Interestingly, pheomelanin has been shown to contribute to the onset and development of melanoma in an ultraviolet-radiation-independent manner through a mechanism of oxidative stress. Eumelanin, on the other hand, is a known antioxidant whose chemical properties seem to shield cells against oxidative damage. To study these pigments in closer detail, nonlinear optical microscopy including coherent anti-Stokes Raman scattering (CARS) was used for the specific visualization and quantification of the relative abundance of pheomelanin and eumelanin within these treatment resistant cell lines. These microscopy toolkits provide a means to monitor changes in pigmentation in a noninvasive and non-destructive manner without the use of exogenous dyes to better understand the molecular basis of treatment resistance.

  12. Clinicopathological characteristics, diagnosis and treatment of melanoma in Serbia--the Melanoma Focus Study.

    PubMed

    Kandolf-Sekulović, Lidija; Babović, Nada; Jokić, Neven; Nikolin, Borislava; Nikolić, Dejan; Janjić, Zlata; Mijugković, Zeljko; Rajović, Milica; Novaković, Marijan; Pejcić, Ivica; Kovaević, Predrag; Mihajlović, Dragan; Roganović, Tatjana; Ferenc, Vicko; Nikolić, Jelena; Marinković, Marija; Bizetić, Zorana

    2015-04-01

    Treatment options for metastatic melanoma in Serbia are limited due to the lack of newly approved biologic agents and the lack of clinical studies. Also, there is a paucity of data regarding the treatment approaches in different tertiary centers and efficacy of available chemotherapy protocols. The aim of this study was to obtain more detailed data about treatment protocols in Serbia based on structured survey in tertiary oncology centers. Data about the melanoma patients treated in 2011 were analyzed from hospital databases in 6 referent oncology centers in Serbia, based on the structured survey, with the focus on metastatic melanoma patients (unresectable stage IIIC and IV). A total of 986 (79-315 in different centers) patients were treated, with 320 (32.45%) newly diagnosed patients. There were 317 patients in stage IIIC/IV, 77/317 aged < 50 years. At the time of diagnosis 47.3% of patients were < 60 years of age (24.2% < 40 years, 23% 50-59 years, 52.6% > 60 years). At initial diagnosis 12.5% of patients were in stage III and 4.5% in stage IV. The most common type was superficial spreading melanoma (50-660), followed by nodular melanoma (23.5-50%). Apart from the regional and distant lymph node metastases, the most frequent organs involved in stage IV disease were distant skin and soft tissues (12-55%), lungs (19-55.5%), liver (10-60%), and bones (3-10%). The first line therapy in stage IV metastatic melanoma was dacarbazine (DTIC) dimethyl-triazenoimidozole-carboxamide in 61-93% of the patients, while the second line varied between the centers. Disease control (complete response + partial response + stable disease) was achieved in 25.7% of the patients treated with the first line chemotherapy and 23.1% of the patients treated with the second line therapy, but the duration of response was short, in first-line therapy 6.66 +/- 3.36 months (median 6.75 months). More than 90% of patients were treated outside the clinical trials. Based on this survey, there is a

  13. Acquired IFNγ resistance impairs anti-tumor immunity and gives rise to T-cell-resistant melanoma lesions

    PubMed Central

    Sucker, Antje; Zhao, Fang; Pieper, Natalia; Heeke, Christina; Maltaner, Raffaela; Stadtler, Nadine; Real, Birgit; Bielefeld, Nicola; Howe, Sebastian; Weide, Benjamin; Gutzmer, Ralf; Utikal, Jochen; Loquai, Carmen; Gogas, Helen; Klein-Hitpass, Ludger; Zeschnigk, Michael; Westendorf, Astrid M.; Trilling, Mirko; Horn, Susanne; Schilling, Bastian; Schadendorf, Dirk; Griewank, Klaus G.; Paschen, Annette

    2017-01-01

    Melanoma treatment has been revolutionized by antibody-based immunotherapies. IFNγ secretion by CD8+ T cells is critical for therapy efficacy having anti-proliferative and pro-apoptotic effects on tumour cells. Our study demonstrates a genetic evolution of IFNγ resistance in different melanoma patient models. Chromosomal alterations and subsequent inactivating mutations in genes of the IFNγ signalling cascade, most often JAK1 or JAK2, protect melanoma cells from anti-tumour IFNγ activity. JAK1/2 mutants further evolve into T-cell-resistant HLA class I-negative lesions with genes involved in antigen presentation silenced and no longer inducible by IFNγ. Allelic JAK1/2 losses predisposing to IFNγ resistance development are frequent in melanoma. Subclones harbouring inactivating mutations emerge under various immunotherapies but are also detectable in pre-treatment biopsies. Our data demonstrate that JAK1/2 deficiency protects melanoma from anti-tumour IFNγ activity and results in T-cell-resistant HLA class I-negative lesions. Screening for mechanisms of IFNγ resistance should be considered in therapeutic decision-making. PMID:28561041

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

    PubMed

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

    2018-01-01

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

  15. Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET

    PubMed Central

    Peinado, Héctor; Alečković, Maša; Lavotshkin, Simon; Matei, Irina; Costa-Silva, Bruno; Moreno-Bueno, Gema; Hergueta-Redondo, Marta; Williams, Caitlin; García-Santos, Guillermo; Nitadori-Hoshino, Ayuko; Hoffman, Caitlin; Badal, Karen; Garcia, Benjamin A.; Callahan, Margaret K.; Yuan, Jianda; Martins, Vilma R.; Skog, Johan; Kaplan, Rosandra N.; Brady, Mary S.; Wolchok, Jedd D.; Chapman, Paul B.; Kang, Yibin; Bromberg, Jacqueline; Lyden, David

    2013-01-01

    Tumor-derived exosomes are emerging mediators of tumorigenesis with tissue-specific addresses and messages. We explored the function of melanoma-derived exosomes in the formation of primary tumor and metastases in mouse and human subjects. Exosomes from highly metastatic melanoma increased the metastatic behavior of primary tumors by permanently “educating” bone marrow (BM) progenitors via the MET receptor. Melanoma-derived exosomes also induced vascular leakiness at pre-metastatic sites, and reprogrammed BM progenitors towards a c-Kit+Tie2+Met+ pro-vasculogenic phenotype. Reducing Met expression in exosomes diminished the pro-metastatic behavior of BM cells. Importantly, MET expression was elevated in circulating CD45−C-KITlow/+TIE2+ BM progenitors from metastatic melanoma subjects. RAB1a, RAB5b, RAB7, and RAB27a were highly expressed in melanoma cells and Rab27a RNA interference decreased exosome production, preventing BM education, tumor growth and metastasis. Finally, we identified an exosome-specific “melanoma signature” with prognostic and therapeutic potential, comprised of TYRP2, VLA-4, HSP70, an HSP90 isoform and the MET oncoprotein. PMID:22635005

  16. ATG5 mediates a positive feedback loop between Wnt signaling and autophagy in melanoma

    PubMed Central

    Ndoye, Abibatou; Budina-Kolomets, Anna; Kugel, Curtis H.; Webster, Marie; Kaur, Amanpreet; Behera, Reeti; Rebecca, Vito; Li, Ling; Brafford, Patricia; Liu, Qin; Gopal, Y.N. Vashisht; Davies, Michael A.; Mills, Gordon B.; Xu, Xiaowei; Wu, Hong; Herlyn, Meenhard; Nicastri, Michael; Winkler, Jeffrey; Soengas, Maria S.; Amaravadi, Ravi; Murphy, Maureen; Weeraratna, Ashani T.

    2017-01-01

    Autophagy mediates resistance to various anticancer agents. In melanoma, resistance to targeted therapy has been linked to expression of Wnt5A, an intrinsic inhibitor of β-catenin, which also promotes invasion. In this study, we assessed the interplay between Wnt5A and autophagy by combining expression studies in human clinical biopsies with functional analyses in cell lines and mouse models. Melanoma cells with high Wnt5A and low β-catenin displayed increased basal autophagy. Genetic blockade of autophagy revealed an unexpected feedback loop whereby knocking down the autophagy factor ATG5 in Wnt5Ahigh cells decreased Wnt5A and increased β-catenin. To define the physiological relevance of this loop, melanoma cells with different Wnt status were treated in vitro and in vivo with the potent lysosomotropic compound Lys05. Wnt5Ahigh cells were less sensitive to Lys05 and could be reverted by inducing β-catenin activity. Our results suggest the efficacy of autophagy inhibitors might be improved by taking the Wnt signature of melanoma cells into account. PMID:28887323

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

    PubMed

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

    2017-04-01

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

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

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

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

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

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

    PubMed

    Landen, Jaren W; Lang, Roland; McMahon, Steve J; Rusan, Nasser M; Yvon, Anne-Marie; Adams, Ashley W; Sorcinelli, Mia D; Campbell, Ross; Bonaccorsi, Paola; Ansel, John C; Archer, David R; Wadsworth, Patricia; Armstrong, Cheryl A; Joshi, Harish C

    2002-07-15

    Cellular microtubules, polymers of tubulin, alternate relentlessly between phases of growth and shortening. We now show that noscapine, a tubulin-binding agent, increases the time that cellular microtubules spend idle in a paused state. As a result, most mammalian cell types observed arrest in mitosis in the presence of noscapine. We demonstrate that noscapine-treated murine melanoma B16LS9 cells do not arrest in mitosis but rather become polyploid followed by cell death, whereas primary melanocytes reversibly arrest in mitosis and resume a normal cell cycle after noscapine removal. Furthermore, in a syngeneic murine model of established s.c. melanoma, noscapine treatment resulted in an 85% inhibition of tumor volume on day 17 when delivered by gavage compared with untreated animals (P melanoma progression by 83% on day 18 when delivered in drinking water (P melanoma cells through alterations in microtubule dynamics, with no detected toxicity to the host. Consequently, noscapine could be a valuable chemotherapeutic agent, alone or in combination, for the treatment of advanced melanoma.

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

    PubMed

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

    2017-09-15

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

  1. PROTEIN KINASE C δ IS A THERAPEUTIC TARGET IN MALIGNANT MELANOMA WITH NRAS MUTATION

    PubMed Central

    Takashima, Asami; English, Brandon; Chen, Zhihong; Cao, Juxiang; Cui, Rutao; Williams, Robert M.; Faller, Douglas V.

    2014-01-01

    NRAS is the second most frequently mutated gene in melanoma. Previous reports have demonstrated the sensitivity of cancer cell lines carrying KRAS mutations to apoptosis initiated by inhibition of protein kinase C delta (PKCδ). Here, we report that PKCδ inhibition is cytotoxic in melanomas with primary NRAS mutations. Novel small-molecule inhibitors of PKCδ were designed as chimeric hybrids of two naturally-occurring PKCδ inhibitors, staurosporine and rottlerin. The specific hypothesis interrogated and validated is that combining two domains of two naturally-occurring PKCδ inhibitors into a chimeric or hybrid structure retains biochemical and biological activity, and improves PKCδ isozyme selectivity. We have devised a potentially general synthetic protocol to make these chimeric species using Molander trifluorborate coupling chemistry. Inhibition of PKCδ, by siRNA or small molecule inhibitors, suppressed the growth of multiple melanoma cell lines carrying NRAS mutations, mediated via caspase-dependent apoptosis. Following PKCδ inhibition, the stress-responsive JNK pathway was activated, leading to the activation of H2AX. Consistent with recent reports on the apoptotic role of phospho-H2AX, knockdown of H2AX prior to PKCδ inhibition mitigated the induction of caspase-dependent apoptosis. Furthermore, PKCδ inhibition effectively induced cytotoxicity in BRAF-mutant melanoma cell lines that had evolved resistance to a BRAF inhibitor, suggesting the potential clinical application of targeting PKCδ in patients who have relapsed following treatment with BRAF inhibitors. Taken together, the present work demonstrates that inhibition of PKCδ by novel small molecule inhibitors causes caspase-dependent apoptosis mediated via the JNK-H2AX pathway in melanomas with NRAS mutations or BRAF inhibitor-resistance. PMID:24506253

  2. B-Raf inhibitor vemurafenib in combination with temozolomide and fotemustine in the killing response of malignant melanoma cells.

    PubMed

    Roos, Wynand P; Quiros, Steve; Krumm, Andrea; Merz, Stephanie; Switzeny, Olivier Jérôme; Christmann, Markus; Loquai, Carmen; Kaina, Bernd

    2014-12-30

    In the treatment of metastatic melanoma, a highly therapy-refractory cancer, alkylating agents are used and, for the subgroup of BRAFV600E cancers, the B-Raf inhibitor vemurafenib. Although vemurafenib is initially beneficial, development of drug resistance occurs leading to tumor relapse, which necessitates the requirement for combined or sequential therapy with other drugs, including genotoxic alkylating agents. This leads to the question whether vemurafenib and alkylating agents act synergistically and whether chronic vemurafenib treatment alters the melanoma cell response to alkylating agents. Here we show that a) BRAFV600E melanoma cells are killed by vemurafenib, driving apoptosis, b) BRAFV600E melanoma cells are neither more resistant nor sensitive to temozolomide/fotemustine than non-mutant cells, c) combined treatment with vemurafenib plus temozolomide or fotemustine has an additive effect on cell kill, d) acquired vemurafenib resistance of BRAFV600E melanoma cells does not affect MGMT, MSH2, MSH6, PMS2 and MLH1, nor does it affect the resistance to temozolomide and fotemustine, e) metastatic melanoma biopsies obtained from patients prior to and after vemurafenib treatment did not show a change in the MGMT promoter methylation status and MGMT expression level. The data suggest that consecutive treatment with vemurafenib and alkylating drugs is a reasonable strategy for metastatic melanoma treatment.

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

    PubMed

    Kuwajima, Akiko; Iwashita, Jun; Murata, Jun; Abe, Tatsuya

    2007-01-01

    Histone deacetylase (HDAC) inhibitors have anticancer effects. Their effects on expression of cell adhesion molecules might be related to their effects on tumor cell invasion. Murine B16-BL6 cells were treated with the HDAC inhibitors, butyrate or trichostatin A. Melanoma cell invasion of the artificial basement membrane, Matrigel, was examined by Transwell chamber assay. Butyrate as well as trichostatin A inhibited the cell growth mainly by arresting the cell cycle. The cell invasion of Matrigel was inhibited by butyrate and trichostatin A. The butyrate treatment increased the cell-cell aggregation, although neither E-cadherin nor N-cadherin mRNA were up-regulated. Both mRNA expression and protein levels of the immunoglobulin superfamily cell adhesion molecules, Mel-CAM and L1-CAM, were increased in the butyrate-treated cells. The HDAC inhibitor butyrate blocked the B16-BL6 melanoma cell invasion of Matrigel, although it increased the expression of Mel-CAM and L1-CAM which are important to the metastatic potential.

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2017-01-01

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

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

    PubMed

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

    2007-03-01

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

  7. Tyrosinase overexpression promotes ATM-dependent p53 phosphorylation by quercetin and sensitizes melanoma cells to dacarbazine.

    PubMed

    Thangasamy, Thilakavathy; Sittadjody, Sivanandane; Limesand, Kirsten H; Burd, Randy

    2008-01-01

    Dacarbazine (DTIC) has been used for the treatment of melanoma for decades. However, monotherapy with this chemotherapeutic agent results only in moderate response rates. To improve tumor response to DTIC current clinical trials in melanoma focus on combining a novel targeted agent with chemotherapy. Here, we demonstrate that tyrosinase which is commonly overexpressed in melanoma activates the bioflavonoid quercetin (Qct) and promotes an ataxia telangiectasia mutated (ATM)-dependent DNA damage response. This response sensitizes melanoma cells that overexpress tyrosinase to DTIC. In DB-1 melanoma cells that overexpress tyrosinase (Tyr(+) cells), the threshold for phosphorylation of ATM and p53 at serine 15 was observed at a low dose of Qct (25 microM) when compared to the mock transfected pcDNA3 cells, which required a higher dose (75 microM). Both pcDNA3 and Tyr(+) DB-1 cells demonstrated similar increases in phosphorylation of p53 at other serine sites, but in the Tyr(+) cells, DNApk expression was found to be reduced compared to control cells, indicating a shift towards an ATM-mediated response. The DB-1 control cells were resistant to DTIC, but were sensitized to apoptosis with high dose Qct, while Tyr(+) cells were sensitized to DTIC with low or high dose Qct. Qct also sensitized SK Mel 5 (p53 wildtype) and 28 (p53 mutant) cells to DTIC. However, when SK Mel 5 cells were transiently transfected with tyrosinase and treated with Qct plus DTIC, SK Mel 5 cells demonstrated a more than additive induction of apoptosis. Therefore, this study demonstrates that tyrosinase overexpression promotes an ATM-dependent p53 phosphorylation by Qct treatment and sensitizes melanoma cells to dacarbazine. In conclusion, these results suggest that Qct or Qct analogues may significantly improve DTIC response rates in tumors that express tyrosinase.

  8. Tyrosinase Overexpression Promotes ATM-Dependent p53 Phosphorylation by Quercetin and Sensitizes Melanoma Cells to Dacarbazine

    PubMed Central

    Thangasamy, Thilakavathy; Sittadjody, Sivanandane; H. Limesand, Kirsten; Burd, Randy

    2008-01-01

    Dacarbazine (DTIC) has been used for the treatment of melanoma for decades. However, monotherapy with this chemotherapeutic agent results only in moderate response rates. To improve tumor response to DTIC current clinical trials in melanoma focus on combining a novel targeted agent with chemotherapy. Here, we demonstrate that tyrosinase which is commonly overexpressed in melanoma activates the bioflavonoid quercetin (Qct) and promotes an ataxia telangiectasia mutated (ATM)-dependent DNA damage response. This response sensitizes melanoma cells that overexpress tyrosinase to DTIC. In DB-1 melanoma cells that overexpress tyrosinase (Tyr cells), the threshold for phosphorylation of ATM and p53 at serine 15 was observed at a low dose of Qct (25 μM) when compared to the mock transfected pcDNA3 cells, which required a higher dose (75 μM). Both pcDNA3 and Tyr DB-1 cells demonstrated similar increases in phosphorylation of p53 at other serine sites, but in the Tyr cells, DNApk expression was found to be reduced compared to control cells, indicating a shift towards an ATM-mediated response. The DB-1 control cells were resistant to DTIC, but were sensitized to apoptosis with high dose Qct, while Tyr cells were sensitized to DTIC with low or high dose Qct. Qct also sensitized SK Mel 5 (p53 wildtype) and 28 (p53 mutant) cells to DTIC. However, when SK Mel 5 cells were transiently transfected with tyrosinase and treated with Qct plus DTIC, SK Mel 5 cells demonstrated a more than additive induction of apoptosis. Therefore, this study demonstrates that tyrosinase overexpression promotes an ATM-dependent p53 phosphorylation by Qct treatment and sensitizes melanoma cells to dacarbazine. In conclusion, these results suggest that Qct or Qct analogues may significantly improve DTIC response rates in tumors that express tyrosinase. PMID:18791269

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

    PubMed

    Andoh, Tsugunobu; Shinohara, Akira; Kuraishi, Yasushi

    2017-02-01

    Our previous study showed that the μ-opioid receptor agonist fentanyl citrate inhibits endothelin-1-and bradykinin-mediated pain responses in mice orthotopically inoculated with melanoma cells. We also demonstrated that bradykinin induces endothelin-1 secretion in melanoma cells. However, the analgesic mechanisms of fentanyl citrate remain unclear. Thus, the present study was conducted to determine whether fentanyl citrate affects bradykinin-induced endothelin-1 secretion in B16-BL6 melanoma cells. The amount of endothelin-1 in the culture medium was measured using an enzyme immunoassay. The expression of endothelin-1, kinin B 2 receptors, and μ-opioid receptors in B16-BL/6 melanoma cells was determined using immunocytochemistry. Fentanyl citrate inhibited bradykinin-induced endothelin-1 secretion. The inhibitory effect of fentanyl citrate on the secretion of endothelin-1 was attenuated by the μ-opioid receptor antagonist naloxone methiodide. The immunoreactivities of endothelin-1, kinin B 2 receptors, and μ-opioid receptors in B16-BL6 melanoma cells were observed. These results suggest that fentanyl citrate regulates bradykinin-induced endothelin-1 secretion through μ-opioid receptors in melanoma cells. Copyright © 2016 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

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

    PubMed

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

    2011-04-01

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

  11. Limited genomic heterogeneity of circulating melanoma cells in advanced stage patients

    NASA Astrophysics Data System (ADS)

    Ruiz, Carmen; Li, Julia; Luttgen, Madelyn S.; Kolatkar, Anand; Kendall, Jude T.; Flores, Edna; Topp, Zheng; Samlowski, Wolfram E.; McClay, Edward; Bethel, Kelly; Ferrone, Soldano; Hicks, James; Kuhn, Peter

    2015-02-01

    Purpose. Circulating melanoma cells (CMCs) constitute a potentially important representation of time-resolved tumor biology in patients. To date, genomic characterization of CMCs has been limited due to the lack of a robust methodology capable of identifying them in a format suitable for downstream characterization. Here, we have developed a methodology to detect intact CMCs that enables phenotypic, morphometric and genomic analysis at the single cell level. Experimental design. Blood samples from 40 metastatic melanoma patients and 10 normal blood donors were prospectively collected. A panel of 7 chondroitin sulfate proteoglycan 4 (CSPG4)-specific monoclonal antibodies (mAbs) was used to immunocytochemically label CMCs. Detection was performed by automated digital fluorescence microscopy and multi-parametric computational analysis. Individual CMCs were captured by micromanipulation for whole genome amplification and copy number variation (CNV) analysis. Results. Based on CSPG4 expression and nuclear size, 1-250 CMCs were detected in 22 (55%) of 40 metastatic melanoma patients (0.5-371.5 CMCs ml-1). Morphometric analysis revealed that CMCs have a broad spectrum of morphologies and sizes but exhibit a relatively homogeneous nuclear size that was on average 1.5-fold larger than that of surrounding PBMCs. CNV analysis of single CMCs identified deletions of CDKN2A and PTEN, and amplification(s) of TERT, BRAF, KRAS and MDM2. Furthermore, novel chromosomal amplifications in chr12, 17 and 19 were also found. Conclusions. Our findings show that CSPG4 expressing CMCs can be found in the majority of advanced melanoma patients. High content analysis of this cell population may contribute to the design of effective personalized therapies in patients with melanoma.

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

    PubMed

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

    2013-04-01

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

  13. Effect of the oncolytic ECHO-7 virus Rigvir® on the viability of cell lines of human origin in vitro.

    PubMed

    Tilgase, Andra; Patetko, Liene; Blāķe, Ilze; Ramata-Stunda, Anna; Borodušķis, Mārtiņš; Alberts, Pēteris

    2018-01-01

    Background: The role of oncolytic viruses in cancer treatment is increasingly studied. The first oncolytic virus (Rigvir®, ECHO-7) was registered in Latvia over a decade ago. In a recent retrospective study Rigvir® decreased mortality 4.39-6.57-fold in stage IB-IIC melanoma patients. The aims of the present study are to test the effect of Rigvir® on cell line viability in vitro and to visualize the cellular presence of Rigvir® by immunocytochemistry. Methods: The cytolytic effect of Rigvir® on the viability of FM-9, RD, AGS, A549, HDFa, HPAF‑II, MSC, MCF7, HaCaT, and Sk-Mel-28 cell lines was measured using live cell imaging. PBMC viability was measured using flow cytometry. The presence of ECHO-7 virus was visualized using immunocytochemistry. Statistical difference between treatment groups was calculated using two-way ANOVA. Results: Rigvir® (10%, volume/volume) reduced cell viability in FM-9, RD, AGS, A549, HDFa, HPAF‑II and MSC cell lines by 67-100%. HaCaT cell viability was partly affected while Rigvir® had no effect on MCF7, Sk-Mel-28 and PBMC viability. Detection of ECHO-7 by immunocytochemistry in FM-9, RD, AGS, A549, HDFa, HPAF-II and Sk-Mel-28 cell lines suggests that the presence of Rigvir® in the cells preceded or coincided with the time of reduction of cell viability. Rigvir® (10%) had no effect on live PBMC count. Conclusions: The results suggest that Rigvir® in vitro reduces the viability of cells of human melanoma, rhabdomyosarcoma, gastric adenocarcinoma, lung carcinoma, pancreas adenocarcinoma but not in PBMC. The presence of Rigvir® in the sensitive cells was confirmed using anti-ECHO-7 antibodies. The present results suggest that a mechanism of action for the clinical benefit of Rigvir® is its cytolytic properties. The present results suggest that the effect of Rigvir® could be tested in other cancers besides melanoma. Further studies of possible Rigvir® entry receptors are needed.

  14. Apoptosis and injuries of heavy ion beam and x-ray radiation on malignant melanoma cell.

    PubMed

    Qin, Jin; Li, Sha; Zhang, Chao; Gao, Dong-Wei; Li, Qiang; Zhang, Hong; Jin, Xiao-Dong; Liu, Yang

    2017-05-01

    This study aims to investigate the influence of high linear energy transfer (LET) heavy ion ( 12 C 6+ ) and low LET X-ray radiation on apoptosis and related proteins of malignant melanoma on tumor-bearing mice under the same physical dosage. C57BL/6 J mice were burdened by tumors and randomized into three groups. These mice received heavy ion ( 12 C 6+ ) and X-ray radiation under the same physical dosage, respectively; their weight and tumor volumes were measured every three days post-radiation. After 30 days, these mice were sacrificed. Then, median survival time was calculated and tumors on mice were proliferated. In addition, immunohistochemistry was carried out for apoptosis-related proteins to reflect the expression level. After tumor-bearing mice were radiated to heavy ion, median survival time improved and tumor volume significantly decreased in conjunction with the upregulated expression of pro-apoptosis factors, Bax and cytochrome C, and the downregulated expression of apoptosis-profilin (Bcl-2, Survivin) and proliferation-related proteins (proliferating cell nuclear antigen). The results indicated that radiation can promote the apoptosis of malignant melanoma cells and inhibit their proliferation. This case was more suitable for heavy ion ( 12 C 6+ ). High LET heavy ion ( 12 C 6+ ) radiation could significantly improve the killing ability for malignant melanoma cells by inducing apoptosis in tumor cells and inhibiting their proliferation. These results demonstrated that heavy ion ( 12 C 6+ ) presented special advantages in terms of treating malignant melanoma. Impact statement Malignant melanoma is a malignant skin tumor derived from melanin cells, which has a high malignant degree and high fatality rate. In this study, proliferating cell nuclear antigen (PCNA) can induce the apoptosis of malignant melanoma cells and inhibit its proliferation, and its induction effect on apoptosis is significantly higher than low LET X-ray; hence, it is expected to

  15. Comparison of a treatment strategy combining CCI-779 plus DTIC versus DTIC monotreatment in human melanoma in SCID mice.

    PubMed

    Thallinger, Christiane; Werzowa, Johannes; Poeppl, Wolfgang; Kovar, Florian M; Pratscher, Barbara; Valent, Peter; Quehenberger, Peter; Joukhadar, Christian

    2007-10-01

    This study compares the antineoplastic potential of a novel treatment strategy combining cell cycle inhibitor-779 (CCI-779) plus dacarbazine (DTIC) versus DTIC monotreatment, the current chemotherapeutic mainstay in combating metastatic melanoma. A controlled four-group parallel study design comprising 24-40 mice per tumor cell line was used in a severe combined immunodeficiency (SCID)-mouse xenotransplantation model. SCID mice were injected with 518A2, Mel-JUSO, or 607B human melanoma cells. After they developed tumors, mice received daily CCI-779 or solvent over 14 days. From treatment day 4-8 mice were additionally injected with DTIC or saline. Treatment with CCI-779 plus DTIC was superior to single agent DTIC in two out of three cell lines (P<0.05). The tumor weight reduction was 44+/-17 and 61+/-6% compared with DTIC monotreatment in Mel-JUSO and 607B melanomas, respectively (P<0.05). In contrast, in 518A2 xenotransplants, CCI-779 plus DTIC treatment was as effective as DTIC monotreatment. CCI-779 monotherapy exerted no statistically significant antitumor effect. Collectively, these data indicate that CCI-779 has the potential to increase the chemotherapeutic efficacy, as the combination of CCI-779 plus DTIC proved to be more efficacious compared to DTIC monotherapy in two out of three melanoma cell lines in vivo.

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

    PubMed

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

    2008-11-01

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

  17. miR-425 inhibits melanoma metastasis through repression of PI3K-Akt pathway by targeting IGF-1.

    PubMed

    Liu, Pei; Hu, Yaotian; Ma, Ling; Du, Min; Xia, Lin; Hu, Zhensheng

    2015-10-01

    miR-425 is a potential tumor suppressor in cancer, but its role in melanoma is still unknown. We aim to investigate miR-425 expression in melanoma tissues and cell lines. Next, cell proliferation, cell cycle, apoptosis and metastasis will be studied using lentivirus-mediated gain-of-function studies. The predicted results are stable miR-425 inhibits cell proliferation and metastasis and induced cell apoptosis. It is predicted that IGF-1 is a potential target gene of miR-495 by bioinformatics analysis. Then luciferase assay analysis identifies IGF-1 as a new direct target gene of miR-425 and miR-425 inhibits melanoma cancer progression via IGF-1. Collectively, our findings suggested that miR-425 may function as a tumor suppressor in melanoma by targeting IGF-1. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  18. Deltex-3-like (DTX3L) stimulates metastasis of melanoma through FAK/PI3K/AKT but not MEK/ERK pathway

    PubMed Central

    Thang, Nguyen Dinh; Yajima, Ichiro; Kumasaka, Mayuko Y.; Iida, Machiko; Suzuki, Tamio; Kato, Masashi

    2015-01-01

    Deltex-3-like (DTX3L), an E3 ligase, is a member of the Deltex (DTX) family and is also called B-lymphoma and BAL-associated protein (BBAP). Previously, we established RFP/RET-transgenic mice, in which systemic hyperpigmented skin, benign melanocytic tumor(s) and melanoma(s) develop stepwise. Here we showed that levels of Dtx3l/DTX3L in spontaneous melanoma in RFP/RET-transgenic mice and human melanoma cell lines were significantly higher than those in benign melanocytic cells and primarily cultured normal human epithelial melanocytes, respectively. Immunohistochemical analysis of human tissues showed that more than 80% of the melanomas highly expressed DTX3L. Activity of FAK/PI3K/AKT signaling, but not that of MEK/ERK signaling, was decreased in Dtx3l/DTX3L-depleted murine and human melanoma cells. In summary, we demonstrated not only increased DTX3L level in melanoma cells but also DTX3L-mediated regulation of invasion and metastasis in melanoma through FAK/PI3K/AKT but not MEK/ERK signaling. Our analysis in human BRAFV600E inhibitor-resistant melanoma cells showed about 80% decreased invasion in the DTX3L-depleted cells compared to that in the DTX3L-intact cells. Thus, DTX3L is clinically a potential therapeutic target as well as a potential biomarker for melanoma. PMID:26033450

  19. In vivo label-free photoacoustic flow cytography and on-the-spot laser killing of single circulating melanoma cells

    NASA Astrophysics Data System (ADS)

    He, Yun; Wang, Lidai; Shi, Junhui; Yao, Junjie; Li, Lei; Zhang, Ruiying; Huang, Chih-Hsien; Zou, Jun; Wang, Lihong V.

    2016-12-01

    Metastasis causes as many as 90% of cancer-related deaths, especially for the deadliest skin cancer, melanoma. Since hematogenous dissemination of circulating tumor cells is the major route of metastasis, detection and destruction of circulating tumor cells are vital for impeding metastasis and improving patient prognosis. Exploiting the exquisite intrinsic optical absorption contrast of circulating melanoma cells, we developed dual-wavelength photoacoustic flow cytography coupled with a nanosecond-pulsed melanoma-specific laser therapy mechanism. We have successfully achieved in vivo label-free imaging of rare single circulating melanoma cells in both arteries and veins of mice. Further, the photoacoustic signal from a circulating melanoma cell immediately hardware-triggers a lethal pinpoint laser irradiation to kill it on the spot in a thermally confined manner without causing collateral damage. A pseudo-therapy study including both in vivo and in vitro experiments demonstrated the performance and the potential clinical value of our method, which can facilitate early treatment of metastasis by clearing circulating tumor cells from vasculature.

  20. Simulated microgravity reduces mRNA levels of multidrug resistance genes 4 and 5 in non-metastatic human melanoma cells

    NASA Astrophysics Data System (ADS)

    Eiermann, Peter; Tsiockas, Wasiliki; Hauslage, Jens; Hemmersbach, Ruth; Gerzer, Rupert; Ivanova, Krassimira

    Multidrug resistance proteins (MRP) are members of the ATP-binding cassette transporter superfamily that are able to export a large variety of substances into the extracellular space in-cluding nucleoside and nucleotide base analogs used in antiviral and anticancer therapy. MRP4 and 5 (MRP4/5) particularly transport cyclic nucleotides, e.g. guanosine 3',5'-cyclic monophos-phate (cGMP). The second messenger cGMP, which is synthesized by the catalytic activity of the guanylyl cyclase (GC), plays an import role in vasodilatation, smooth muscle relaxation, and nitric oxide (NO)-induced perturbation of melanocyte-extracellular matrix interactions. In previous studies we have reported that different GC isoforms are responsible for cGMP synthe-sis in melanocytic cells. Normal human melanocytes and non-metastatic melanoma cell lines predominantly express the NO-sensitive soluble GC isoform (sGC), a heterodimeric protein consisting of α and β subunits. Metastatic melanoma cells lack the expression of the β sub-unit and show up-regulated activities of the particulate isoforms. We have further found that long-term exposure to hypergravity (5 g for 24 h) induced an increased cGMP export in normal human melanocytes, and non-metastatic, but not in metastatic human melanoma cells as a re-sult of up-regulated MRP4/5 expression. The aim of the present study is to investigate whether simulated microgravity may also alter the expression of MRP4/5 in non-metastatic melanoma cells. Experiments were performed using a fast-rotating clinostat (60 rpm) with one rotation axis. The non-metastatic 1F6 melanoma cells were exposed to simulated microgravity (up to 1.21x10-2 g) for 24 h. The mRNA analyses were performed by a relative calibrator-normalized and efficiency corrected quantitative polymerase chain reaction (Light Cycler R , Roche). Our data show a reduced expression of approximately 35% for MRP4 and of 50% for MRP5 in simulated microgravity in comparison to 1 g controls. Also, the

  1. Response of transformed and normal mouse cell lines to anti-melanin compounds, hyperthermia, and radiation.

    PubMed

    Raaphorst, G P; Azzam, E I

    1992-02-01

    Five cell lines (one parental, two transformed melanin producing, and two transformed non-melanin producing) were evaluated for the responses to 2- and 4-hydroxyanisole (2HA, 4HA) alone or combined with hyperthermia or radiation. All cells exhibited a non-specific toxic response to the two compounds and the effect was exposure time and concentration dependent and was greater for 4HA compared to 2HA. In addition, the two melanin-producing cell lines were more sensitive, demonstrating specific toxicity to such cell lines. The treatment with either 2HA or 4HA combined with heat and radiation resulted mostly in additive or antagonistic effects, except for one combination of 2HA plus radiation in the melanin-producing R25 cells. Thus, while these compounds may be useful in therapy for pigmented melanomas, combined treatment with radiation is not recommended.

  2. B-Raf inhibitor vemurafenib in combination with temozolomide and fotemustine in the killing response of malignant melanoma cells

    PubMed Central

    Krumm, Andrea; Merz, Stephanie; Switzeny, Olivier Jérôme; Christmann, Markus; Loquai, Carmen; Kaina, Bernd

    2014-01-01

    In the treatment of metastatic melanoma, a highly therapy-refractory cancer, alkylating agents are used and, for the subgroup of BRAFV600E cancers, the B-Raf inhibitor vemurafenib. Although vemurafenib is initially beneficial, development of drug resistance occurs leading to tumor relapse, which necessitates the requirement for combined or sequential therapy with other drugs, including genotoxic alkylating agents. This leads to the question whether vemurafenib and alkylating agents act synergistically and whether chronic vemurafenib treatment alters the melanoma cell response to alkylating agents. Here we show that a) BRAFV600E melanoma cells are killed by vemurafenib, driving apoptosis, b) BRAFV600E melanoma cells are neither more resistant nor sensitive to temozolomide/fotemustine than non-mutant cells, c) combined treatment with vemurafenib plus temozolomide or fotemustine has an additive effect on cell kill, d) acquired vemurafenib resistance of BRAFV600E melanoma cells does not affect MGMT, MSH2, MSH6, PMS2 and MLH1, nor does it affect the resistance to temozolomide and fotemustine, e) metastatic melanoma biopsies obtained from patients prior to and after vemurafenib treatment did not show a change in the MGMT promoter methylation status and MGMT expression level. The data suggest that consecutive treatment with vemurafenib and alkylating drugs is a reasonable strategy for metastatic melanoma treatment. PMID:25557167

  3. Analysis of the miRNA Profiles of Melanoma Exosomes Derived Under Normoxic and Hypoxic Culture Conditions.

    PubMed

    Wozniak, Michal; Peczek, Lukasz; Czernek, Liliana; Düchler, Markus

    2017-12-01

    MicroRNAs (miRNAs) transported in melanoma-derived exosomes function as intercellular messengers supporting tumor survival and progression. Hypoxia increases melanoma phenotypic plasticity, drug resistance, and metastasis. We determined the miRNA profiles in exosomes derived from melanoma cells grown under hypoxic and normoxic conditions by microarray analyses and reverse transcription-polymerase chain reaction (RT-PCR) in order to analyze the potential influence of vesicle-transported miRNAs on cancer-related pathways and transcriptional programs. Despite phenotypical differences of the four cell lines used, their exosomes shared the majority of miRNAs. The levels of three miRNAs were higher in normoxic exosomes, whereas 15 miRNAs were significantly more abundant under hypoxic conditions. Pathway analysis pointed at several cellular processes contributing to proliferation, drug resistance, and modification of the tumor microenvironment, including immunosuppression. The miRNA-expression profiles of exosomes from patient-derived melanoma cells are modified by oxygen concentration and reflect the phenotypic changes of melanoma cells under different growth conditions. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

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

    PubMed

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

    2009-10-03

    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.

  5. MEK Inhibitors in the Treatment of Metastatic Melanoma and Solid Tumors.

    PubMed

    Grimaldi, Antonio M; Simeone, Ester; Festino, Lucia; Vanella, Vito; Strudel, Martina; Ascierto, Paolo A

    2017-12-01

    The mitogen-activated protein kinase (MAPK) cascade is an intracellular signaling pathway involved in the regulation of cellular proliferation and the survival of tumor cells. Several different mutations, involving BRAF or NRAS, exert an oncogenic effect by activating the MAPK pathway, resulting in an increase in cellular proliferation. These mutations have become targets for new therapeutic strategies in melanoma and other cancers. Selective MEK inhibitors have the ability to inhibit growth and induce cell death in BRAF- and NRAS-mutant melanoma cell lines. MEK inhibitor therapy in combination with a BRAF inhibitor is more effective and less toxic than treatment with a BRAF inhibitor alone, and has become the standard of care for patients with BRAF-mutated melanoma. Trametinib was the first MEK inhibitor approved for the treatment of BRAF-mutated metastatic melanoma not previously treated with BRAF inhibitors, and is also approved in combination with the BRAF inhibitor dabrafenib. Furthermore, cobimetinib is another MEK inhibitor approved for the treatment of BRAF-mutated metastatic melanoma in combination with a BRAF inhibitor, vemurafenib. The MEK inhibitor binimetinib in combination with the BRAF inhibitor encorafenib is in clinical development. The addition of an anti-PD-1/PD-L1 agent, such as pembrolizumab, durvalumab or atezolizumab, to combined BRAF and MEK inhibition has shown considerable promise, with several trials ongoing in metastatic melanoma. Binimetinib has also shown efficacy in NRAS-mutated melanoma patients. Future possibilities for MEK inhibitors in advanced melanoma, as well as other solid tumors, include their use in combination with other targeted therapies (e.g. anti-CDK4/6 inhibitors) and/or various immune-modulating antibodies.

  6. The CASC15 long intergenic non-coding RNA locus is involved in melanoma progression and phenotype-switching

    PubMed Central

    Lessard, Laurent; Liu, Michelle; Marzese, Diego M.; Wang, Hongwei; Chong, Kelly; Kawas, Neal; Donovan, Nicholas C; Kiyohara, Eiji; Hsu, Sandy; Nelson, Nellie; Izraely, Sivan; Sagi-Assif, Orit; Witz, Isaac P; Ma, Xiao-Jun; Luo, Yuling; Hoon, Dave SB

    2015-01-01

    In recent years, considerable advances have been made in the characterization of protein-coding alterations involved in the pathogenesis of melanoma. However, despite their growing implication in cancer, little is known about the role of long non-coding RNAs in melanoma progression. We hypothesized that copy number alterations of intergenic non-protein coding domains could help identify long intergenic non-coding RNAs (lincRNAs) associated with metastatic cutaneous melanoma. Among several candidates, our approach uncovered the chromosome 6p22.3 CASC15 lincRNA locus as a frequently gained genomic segment in metastatic melanoma tumors and cell lines. The locus was actively transcribed in metastatic melanoma cells, and up-regulation of CASC15 expression was associated with metastatic progression to brain metastasis in a mouse xenograft model. In clinical specimens, CASC15 levels increased during melanoma progression and were independent predictors of disease recurrence in a cohort of 141 patients with AJCC stage III lymph node metastasis. Moreover, siRNA knockdown experiments revealed that CASC15 regulates melanoma cell phenotype switching between proliferative and invasive states. Accordingly, CASC15 levels correlated with known gene signatures corresponding to melanoma proliferative and invasive phenotypes. These findings support a key role for CASC15 in metastatic melanoma. PMID:26016895

  7. Limbal Stem Cell Preservation During Proton Beam Irradiation for Diffuse Iris Melanoma.

    PubMed

    Singh, Arun D; Dupps, William J; Biscotti, Charles V; Suh, John H; Lathrop, Kira L; Nairn, John P; Shih, Helen

    2017-01-01

    To report the outcome after limbal stem cell preservation during proton beam irradiation for diffuse iris melanoma. This is a single-case report of diffuse iris melanoma that was managed with proton beam radiation (53 Gy), wherein preemptively harvested superior and inferior limbal stem cells before radiation were replaced after irradiation. Regeneration of the palisades of Vogt and the limbal stem cells was documented by an optical coherence tomography-based imaging protocol. At 24 months after radiation therapy, best-corrected visual acuity was 20/25. The cornea was clear without evidence of limbal stem cell dysfunction. Clinical examination (including gonioscopy and ultrasound biomicroscopy [UBM]) was indicative of local control, and systemic surveillance was negative for metastatic disease. At posttransplant (21 months), there were more palisade structures visible in both anterior and posterior regions of the superior and inferior limbus, and the linear presentation of the inferior palisades appears to have regenerated. Diffuse iris melanoma can be managed successfully with proton beam radiation while preserving corneal limbal stem cells by harvesting them before radiation and then replacing them after irradiation. Regeneration of the palisades of Vogt could be documented by an optical coherence tomography-based imaging protocol.

  8. Combined regimen of photodynamic therapy mediated by Gallium phthalocyanine chloride and Metformin enhances anti-melanoma efficacy

    PubMed Central

    Filip, Gabriela Adriana; Olteanu, Diana; Cenariu, Mihai; Tabaran, Flaviu; Ion, Rodica Mariana; Gligor, Lucian; Baldea, Ioana

    2017-01-01

    Background Melanoma therapy is challenging, especially in advanced cases, due to multiple developed tumor defense mechanisms. Photodynamic therapy (PDT) might represent an adjuvant treatment, because of its bimodal action: tumor destruction and immune system awakening. In this study, a combination of PDT mediated by a metal substituted phthalocyanine—Gallium phthalocyanine chloride (GaPc) and Metformin was used against melanoma. The study aimed to: (1) find the anti-melanoma efficacy of GaPc-PDT, (2) assess possible beneficial effects of Metformin addition to PDT, (3) uncover some of the mechanisms underlining cell killing and anti-angiogenic effects. Methods Two human lightly pigmented melanoma cell lines: WM35 and M1/15 subjected to previous Metformin exposure were treated by GaPc-PDT. Cell viability, death mechanism, cytoskeleton alterations, oxidative damage, were assessed by means of colorimetry, flowcytometry, confocal microscopy, spectrophotometry, ELISA, Western Blotting. Results GaPc proved an efficient photosensitizer. Metformin addition enhanced cell killing by mechanisms dependent on the cell line, namely apoptosis in the metastatic M1/15 and necrosis in the radial growth phase, WM35. Cell death mechanism relied on the inhibition of nuclear transcription factor (NF)-κB activation and tumor necrosis factor (TNF)—related apoptosis-inducing ligand (TRAIL) sensitization, leading to TRAIL and TNF-α induced apoptosis. Metformin diminished the anti-angiogenic effect of PDT. Conclusions Metformin addition to GaPc-PDT increased tumor cell killing through enhanced oxidative damage and induction of proapoptotic mechanisms, but altered PDT anti-angiogenic effects. General significance Combination of Metformin and PDT might represent a solution to enhance the efficacy, leading to a potential adjuvant role of PDT in melanoma therapy. PMID:28278159

  9. Combined regimen of photodynamic therapy mediated by Gallium phthalocyanine chloride and Metformin enhances anti-melanoma efficacy.

    PubMed

    Tudor, Diana; Nenu, Iuliana; Filip, Gabriela Adriana; Olteanu, Diana; Cenariu, Mihai; Tabaran, Flaviu; Ion, Rodica Mariana; Gligor, Lucian; Baldea, Ioana

    2017-01-01

    Melanoma therapy is challenging, especially in advanced cases, due to multiple developed tumor defense mechanisms. Photodynamic therapy (PDT) might represent an adjuvant treatment, because of its bimodal action: tumor destruction and immune system awakening. In this study, a combination of PDT mediated by a metal substituted phthalocyanine-Gallium phthalocyanine chloride (GaPc) and Metformin was used against melanoma. The study aimed to: (1) find the anti-melanoma efficacy of GaPc-PDT, (2) assess possible beneficial effects of Metformin addition to PDT, (3) uncover some of the mechanisms underlining cell killing and anti-angiogenic effects. Two human lightly pigmented melanoma cell lines: WM35 and M1/15 subjected to previous Metformin exposure were treated by GaPc-PDT. Cell viability, death mechanism, cytoskeleton alterations, oxidative damage, were assessed by means of colorimetry, flowcytometry, confocal microscopy, spectrophotometry, ELISA, Western Blotting. GaPc proved an efficient photosensitizer. Metformin addition enhanced cell killing by mechanisms dependent on the cell line, namely apoptosis in the metastatic M1/15 and necrosis in the radial growth phase, WM35. Cell death mechanism relied on the inhibition of nuclear transcription factor (NF)-κB activation and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) sensitization, leading to TRAIL and TNF-α induced apoptosis. Metformin diminished the anti-angiogenic effect of PDT. Metformin addition to GaPc-PDT increased tumor cell killing through enhanced oxidative damage and induction of proapoptotic mechanisms, but altered PDT anti-angiogenic effects. Combination of Metformin and PDT might represent a solution to enhance the efficacy, leading to a potential adjuvant role of PDT in melanoma therapy.

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

    PubMed

    Le Coz, Vincent; Zhu, Chaobin; Devocelle, Aurore; Vazquez, Aimé; Boucheix, Claude; Azzi, Sandy; Gallerne, Cindy; Eid, Pierre; Lecourt, Séverine; Giron-Michel, Julien

    2016-12-13

    Melanoma is a particularly virulent human cancer, due to its resistance to conventional treatments and high frequency of metastasis. Melanomas contain a fraction of cells, the melanoma-initiating cells (MICs), responsible for tumor propagation and relapse. Identification of the molecular pathways supporting MICs is, therefore, vital for the development of targeted treatments. One factor produced by melanoma cells and their microenvironment, insulin-like growth factor-1 (IGF- 1), is linked to epithelial-mesenchymal transition (EMT) and stemness features in several cancers.We evaluated the effect of IGF-1 on the phenotype and chemoresistance of B16-F10 cells. IGF-1 inhibition in these cells prevented malignant cell proliferation, migration and invasion, and lung colony formation in immunodeficient mice. IGF-1 downregulation also markedly inhibited EMT, with low levels of ZEB1 and mesenchymal markers (N-cadherin, CD44, CD29, CD105) associated with high levels of E-cadherin and MITF, the major regulator of melanocyte differentiation. IGF-1 inhibition greatly reduced stemness features, including the expression of key stem markers (SOX2, Oct-3/4, CD24 and CD133), and the functional characteristics of MICs (melanosphere formation, aldehyde dehydrogenase activity, side population). These features were associated with a high degree of sensitivity to mitoxantrone treatment.In this study, we deciphered new connections between IGF-1 and stemness features and identified IGF-1 as instrumental for maintaining the MIC phenotype. The IGF1/IGF1-R nexus could be targeted for the development of more efficient anti-melanoma treatments. Blocking the IGF-1 pathway would improve the immune response, decrease the metastatic potential of tumor cells and sensitize melanoma cells to conventional treatments.

  11. RNA Sequencing Analysis Reveals Interactions between Breast Cancer or Melanoma Cells and the Tissue Microenvironment during Brain Metastasis

    PubMed Central

    Hosonaga, Mari; Koya, Ikuko

    2017-01-01

    Metastasis is the main cause of treatment failure and death in cancer patients. Metastasis of tumor cells to the brain occurs frequently in individuals with breast cancer, non–small cell lung cancer, or melanoma. Despite recent advances in our understanding of the causes and in the treatment of primary tumors, the biological and molecular mechanisms underlying the metastasis of cancer cells to the brain have remained unclear. Metastasizing cancer cells interact with their microenvironment in the brain to establish metastases. We have now developed mouse models of brain metastasis based on intracardiac injection of human breast cancer or melanoma cell lines, and we have performed RNA sequencing analysis to identify genes in mouse brain tissue and the human cancer cells whose expression is associated specifically with metastasis. We found that the expressions of the mouse genes Tph2, Sspo, Ptprq, and Pole as well as those of the human genes CXCR4, PLLP, TNFSF4, VCAM1, SLC8A2, and SLC7A11 were upregulated in brain tissue harboring metastases. Further characterization of such genes that contribute to the establishment of brain metastases may provide a basis for the development of new therapeutic strategies and consequent improvement in the prognosis of cancer patients. PMID:28210624

  12. Dissection of stromal and cancer cell-derived signals in melanoma xenografts before and after treatment with DMXAA

    PubMed Central

    Henare, K; Wang, L; Wang, L-Cs; Thomsen, L; Tijono, S; Chen, C-Jj; Winkler, S; Dunbar, P R; Print, C; Ching, L-M

    2012-01-01

    Background: The non-malignant cells of the tumour stroma have a critical role in tumour biology. Studies dissecting the interplay between cancer cells and stromal cells are required to further our understanding of tumour progression and methods of intervention. For proof-of-principle of a multi-modal approach to dissect the differential effects of treatment on cancer cells and stromal cells, we analysed the effects of the stromal-targeting agent 5,6-dimethylxanthenone-4-acetic acid on melanoma xenografts. Methods: Flow cytometry and multi-colour immunofluorescence staining was used to analyse leukocyte numbers in xenografts. Murine-specific and human-specific multiplex cytokine panels were used to quantitate cytokines produced by stromal and melanoma cells, respectively. Human and mouse Affymetrix microarrays were used to separately identify melanoma cell-specific and stromal cell-specific gene expression. Results: 5,6-Dimethylxanthenone-4-acetic acid activated pro-inflammatory signalling pathways and cytokine expression from both stromal and cancer cells, leading to neutrophil accumulation and haemorrhagic necrosis and a delay in tumour re-growth of 26 days in A375 melanoma xenografts. Conclusion: 5,6-Dimethylxanthenone-4-acetic acid and related analogues may potentially have utility in the treatment of melanoma. The experimental platform used allowed distinction between cancer cells and stromal cells and can be applied to investigate other tumour models and anti-cancer agents. PMID:22415295

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

    PubMed

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

    2012-10-01

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

  14. Favorable overall survival in stage III melanoma patients after adjuvant dendritic cell vaccination

    PubMed Central

    Bol, Kalijn F; Aarntzen, Erik H J G; Hout, Florentien E M in 't; Schreibelt, Gerty; Creemers, Jeroen H A; Lesterhuis, W Joost; Gerritsen, Winald R; Grunhagen, Dirk J; Verhoef, Cornelis; Punt, Cornelis J A; Bonenkamp, Johannes J; de Wilt, Johannes H W; Figdor, Carl G; de Vries, I Jolanda M

    2016-01-01

    Melanoma patients with regional metastatic disease are at high risk for recurrence and metastatic disease, despite radical lymph node dissection (RLND). We investigated the immunologic response and clinical outcome to adjuvant dendritic cell (DC) vaccination in melanoma patients with regional metastatic disease who underwent RLND with curative intent. In this retrospective study, 78 melanoma patients with regional lymph node metastasis who underwent RLND received autologous DCs loaded with gp100 and tyrosinase and were analyzed for functional tumor-specific T cell responses in skin-test infiltrating lymphocytes. The study shows that adjuvant DC vaccination in melanoma patients with regional lymph node metastasis is safe and induced functional tumor-specific T cell responses in 71% of the patients. The presence of functional tumor-specific T cells was correlated with a better 2-year overall survival (OS) rate. OS was significantly higher after adjuvant DC vaccination compared to 209 matched controls who underwent RLND without adjuvant DC vaccination, 63.6 mo vs. 31.0 mo (p = 0.018; hazard ratio 0.59; 95%CI 0.42–0.84). Five-year survival rate increased from 38% to 53% (p < 0.01). In summary, in melanoma patients with regional metastatic disease, who are at high risk for recurrence and metastatic disease after RLND, adjuvant DC vaccination is well tolerated. It induced functional tumor-specific immune responses in the majority of patients and these were related to clinical outcome. OS was significantly higher compared to matched controls. A randomized clinical trial is needed to prospectively validate the efficacy of DC vaccination in the adjuvant setting. PMID:26942068

  15. 9-AAA inhibits growth and induces apoptosis in human melanoma A375 and rat prostate adenocarcinoma AT-2 and Mat-LyLu cell lines but does not affect the growth and viability of normal fibroblasts.

    PubMed

    Korohoda, Włodzimierz; Hapek, Anna; Pietrzak, Monika; Ryszawy, Damian; Madeja, Zbigniew

    2016-11-01

    The present study found that, similarly to 5-fluorouracil, low concentrations (1-10 µM) of 9-aminoacridine (9-AAA) inhibited the growth of the two rat prostate cancer AT-2 and Mat-LyLu cell lines and the human melanoma A375 cell line. However, at the same concentrations, 9-AAA had no effect on the growth and apoptosis of normal human skin fibroblasts (HSFs). The differences between the cellular responses of the AT-2 and Mat-LyLu cell lines, which differ in malignancy, were found to be relatively small compared with the differences between normal HSFs and the cancer cell lines. Visible effects on the cell growth and survival of tumor cell lines were observed after 24-48 h of treatment with 9-AAA, and increased over time. The inhibition of cancer cell growth was found to be due to the gradually increasing number of cells dying by apoptosis, which was observed using two methods, direct counting and FlowSight analysis. Simultaneously, cell motile activity decreased to the same degree in cancer and normal cells within the first 8 h of incubation in the presence of 9-AAA. The results presented in the current study suggest that short-lasting tests for potential anticancer substances can be insufficient; which may result in cell type-dependent differences in the responses of cells to tested compounds that act with a delay being overlooked. The observed differences in responses between normal human fibroblasts and cancer cells to 9-AAA show the requirement for additional studies to be performed simultaneously on differently reacting cancer and normal cells, to determine the molecular mechanisms responsible for these differences.

  16. Serum levels of cytoplasmic melanoma-associated antigen at diagnosis may predict clinical relapse in neuroblastoma patients

    PubMed Central

    Corrias, Maria Valeria; Levreri, Isabella; Scaruffi, Paola; Raffaghello, Lizzia; Carlini, Barbara; Bocca, Paola; Prigione, Ignazia; Stigliani, Sara; Amoroso, Loredana; Ferrone, Soldano; Pistoia, Vito

    2012-01-01

    The high molecular weight melanoma-associated antigen (HMW-MAA) and the cytoplasmic melanoma-associated antigen (cyt-MAA/LGALS3BP) are expressed in melanoma. Their serum levels are increased in melanoma patients and correlate with clinical outcome. We investigated whether these molecules can serve as prognostic markers for neuroblastoma (NB) patients. Expression of cyt-MAA and HMW-MAA was evaluated by flow cytometry in NB cell lines, patients’ neuroblasts (FI-NB), and short-term cultures of these latter cells (cNB). LGALS3BP gene expression was evaluated by RT–qPCR on FI-NB, cNB, and primary tumor specimens. Soluble HMW-MAA and cyt-MAA were tested by ELISA. Cyt-MAA and HMW-MAA were expressed in NB cell lines, cNB, and FI-NB samples. LGALS3BP gene expression was higher in primary tumors and cNB than in FI-NB samples. Soluble cyt-MAA, but not HMW-MAA, was detected in NB cell lines and cNBs supernatants. NB patients’ serum levels of both antigens were higher than those of the healthy children. High cyt-MAA serum levels at diagnosis associated with higher incidence of relapse, independently from other known risk factors. In conclusion, both HMW-MAA and cyt-MAA antigens, and LGALS3BP gene, were expressed by NB cell lines and patients’ neuroblasts, and both antigens’ serum levels were increased in NB patients. Elevated serum levels of cyt-MAA at diagnosis correlated with relapse, supporting that cyt-MAA may serve as early serological biomarker to individuate patients at higher risk of relapse that may require a more careful follow-up, after being validated in a larger cohort of patients at different time-points during follow-up. Given its immunogenicity, cyt-MAA may also be a potential target for NB immunotherapy. PMID:21660451

  17. Isolation and detection of circulating tumour cells from metastatic melanoma patients using a slanted spiral microfluidic device.

    PubMed

    Aya-Bonilla, Carlos A; Marsavela, Gabriela; Freeman, James B; Lomma, Chris; Frank, Markus H; Khattak, Muhammad A; Meniawy, Tarek M; Millward, Michael; Warkiani, Majid E; Gray, Elin S; Ziman, Mel

    2017-09-15

    Circulating Tumour Cells (CTCs) are promising cancer biomarkers. Several methods have been developed to isolate CTCs from blood samples. However, the isolation of melanoma CTCs is very challenging as a result of their extraordinary heterogeneity, which has hindered their biological and clinical study. Thus, methods that isolate CTCs based on their physical properties, rather than surface marker expression, such as microfluidic devices, are greatly needed in melanoma. Here, we assessed the ability of the slanted spiral microfluidic device to isolate melanoma CTCs via label-free enrichment. We demonstrated that this device yields recovery rates of spiked melanoma cells of over 80% and 55%, after one or two rounds of enrichment, respectively. Concurrently, a two to three log reduction of white blood cells was achieved with one or two rounds of enrichment, respectively. We characterised the isolated CTCs using multimarker flow cytometry, immunocytochemistry and gene expression. The results demonstrated that CTCs from metastatic melanoma patients were highly heterogeneous and commonly expressed stem-like markers such as PAX3 and ABCB5. The implementation of the slanted microfluidic device for melanoma CTC isolation enables further understanding of the biology of melanoma metastasis for biomarker development and to inform future treatment approaches.

  18. Induction of Melanogenesis by Rapamycin in Human MNT-1 Melanoma Cells

    PubMed Central

    Hah, Young-Sool; Cho, Hee Young; Lim, Tae-Yeon; Park, Dong Hwa; Kim, Hwa Mi; Yoon, Jimi; Kim, Jin Gu; Kim, Chi Yeon

    2012-01-01

    Background Melanogenesis is one of the characteristic parameters of differentiation in melanocytes and melanoma cells. Specific inhibitors of phosphatidylinositol 3-kinase (PI3K), such as wortmannin and LY294002, stimulate melanin production in mouse and in human melanoma cells, suggesting that PI3K and mammalian target of rapamycin (mTOR) might be involved in the regulation of melanogenesis. Objective The involvement of the mTOR pathway in regulating melanogenesis was examined using human MNT-1 melanoma cells, and the effects of the potent inhibitor of mTOR, rapamycin, in the presence or absence of α-melanocyte-stimulating hormone (α-MSH) were evaluated. Methods In cells treated with rapamycin, cell viability, melanin content, and tyrosinase (TYR) activity were measured and compared with untreated controls. Protein levels of TYR, tyrosinase-related protein (TYRP)-1, TYRP-2, and microphthalmia-associated transcription factor (MITF) were also analyzed by Western blot. Results In rapamycin-treated cells, the melanin content increased concomitantly with an elevation in TYR activity, which plays a major role in melanogenesis. There was also an up-regulation of TYR, TYRP-1, and MITF proteins. Combined treatment with rapamycin or wortmannin and α-MSH increased melanogenesis more strongly than α-MSH alone. Conclusion Rapamycin-induced melanin formation may be mediated through the up-regulation of TYR protein and activity. Furthermore, rapamycin and wortmannin, inhibitors of mTOR and PI3K, respectively, have co-stimulatory effects with α-MSH in enhancing melanogenesis in melanocyte cells. PMID:22577264

  19. Induction of melanogenesis by rapamycin in human MNT-1 melanoma cells.

    PubMed

    Hah, Young-Sool; Cho, Hee Young; Lim, Tae-Yeon; Park, Dong Hwa; Kim, Hwa Mi; Yoon, Jimi; Kim, Jin Gu; Kim, Chi Yeon; Yoon, Tae-Jin

    2012-05-01

    Melanogenesis is one of the characteristic parameters of differentiation in melanocytes and melanoma cells. Specific inhibitors of phosphatidylinositol 3-kinase (PI3K), such as wortmannin and LY294002, stimulate melanin production in mouse and in human melanoma cells, suggesting that PI3K and mammalian target of rapamycin (mTOR) might be involved in the regulation of melanogenesis. The involvement of the mTOR pathway in regulating melanogenesis was examined using human MNT-1 melanoma cells, and the effects of the potent inhibitor of mTOR, rapamycin, in the presence or absence of α-melanocyte-stimulating hormone (α-MSH) were evaluated. In cells treated with rapamycin, cell viability, melanin content, and tyrosinase (TYR) activity were measured and compared with untreated controls. Protein levels of TYR, tyrosinase-related protein (TYRP)-1, TYRP-2, and microphthalmia-associated transcription factor (MITF) were also analyzed by Western blot. In rapamycin-treated cells, the melanin content increased concomitantly with an elevation in TYR activity, which plays a major role in melanogenesis. There was also an up-regulation of TYR, TYRP-1, and MITF proteins. Combined treatment with rapamycin or wortmannin and α-MSH increased melanogenesis more strongly than α-MSH alone. Rapamycin-induced melanin formation may be mediated through the up-regulation of TYR protein and activity. Furthermore, rapamycin and wortmannin, inhibitors of mTOR and PI3K, respectively, have co-stimulatory effects with α-MSH in enhancing melanogenesis in melanocyte cells.

  20. At the bedside: adoptive cell therapy for melanoma-clinical development.

    PubMed

    Weber, Jeffrey S

    2014-06-01

    Adoptive cell therapy for melanoma, particularly using TIL, consists of a complex and difficult set of procedures, although it has a strong preclinical background and justification and has been pursued clinically by one small group of investigators over the last 20 years. More recent developments and a better understanding of the molecular basis of the anti-tumor immune response have led to the conduct of clinical trials that use lymphoid depletion with chemotherapy and/or TBI to exploit the favorable immune milieu of homeostatic lymphoid reconstitution during transfer of effector T cells. Improved ways of propagating T cells ex vivo have also simplified and shortened the cell-growth process. Current TIL trials have now been expanded beyond the initial center where it was developed, reproducing excellent objective response rates of 40-50% in previously treated melanoma patients and more importantly, demonstrating that a significant proportion of patients will be alive and free of disease 3-5 years after treatment, raising the possibility that those patients may be cured of their disease. Newer methods for growing the infiltrating T cells using immune-checkpoint antibodies or other agents to condition the tumor before harvest and improved technology to simplify the complex and often cumbersome cell-growth process suggest that this technology may be able to be disseminated to a wide selection of cancer centers and may be a candidate for testing in a randomized Phase III trial to show definitively its benefit in patients with metastatic melanoma. In the accompanying review, the preclinical work that supports the idea of adoptive cell therapy with TIL and expands the concept in promising new ways will be explored. © 2014 Society for Leukocyte Biology.

  1. Xeroderma Pigmentosum Group A Promotes Autophagy to Facilitate Cisplatin Resistance in Melanoma Cells through the Activation of PARP1.

    PubMed

    Ge, Rui; Liu, Lin; Dai, Wei; Zhang, Weigang; Yang, Yuqi; Wang, Huina; Shi, Qiong; Guo, Sen; Yi, Xiuli; Wang, Gang; Gao, Tianwen; Luan, Qi; Li, Chunying

    2016-06-01

    Xeroderma pigmentosum group A (XPA), a key protein in the nucleotide excision repair pathway, has been shown to promote the resistance of tumor cells to chemotherapeutic drugs by facilitating the DNA repair process. However, the role of XPA in the resistance of melanoma to platinum-based drugs like cisplatin is largely unknown. In this study, we initially found that XPA was expressed at higher levels in cisplatin-resistant melanoma cells than in cisplatin-sensitive ones. Furthermore, the knockdown of XPA not only increased cellular apoptosis but also inhibited cisplatin-induced autophagy, which rendered the melanoma cells more sensitive to cisplatin. Moreover, we discovered that the increased XPA in resistant melanoma cells promoted poly(adenosine diphosphate-ribose) polymerase 1 (PARP1) activation and that the inhibition of PARP1 could attenuate the cisplatin-induced autophagy. Finally, we proved that the inhibition of PARP1 and the autophagy process made resistant melanoma cells more susceptible to cisplatin treatment. Our study shows that XPA can promote cell-protective autophagy in a DNA repair-independent manner by enhancing the activation of PARP1 in melanoma cells resistant to cisplatin and that the XPA-PARP1-mediated autophagy process can be targeted to overcome cisplatin resistance in melanoma chemotherapy. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  2. Everolimus selectively targets vemurafenib resistant BRAFV600E melanoma cells adapted to low pH.

    PubMed

    Ruzzolini, Jessica; Peppicelli, Silvia; Andreucci, Elena; Bianchini, Francesca; Margheri, Francesca; Laurenzana, Anna; Fibbi, Gabriella; Pimpinelli, Nicola; Calorini, Lido

    2017-11-01

    Vemurafenib, a BRAF inhibitor, elicits in ∼80% of BRAF V600E -mutant melanoma patients a transient anti-tumor response which precedes the emergence of resistance. We tested whether an acidic tumor microenvironment may favor a BRAF inhibitor resistance. A375M6 BRAF V600E melanoma cells, either exposed for a short period or chronically adapted to an acidic medium, showed traits compatible with an epithelial-mesenchymal transition, reduced proliferation and high resistance to apoptosis. Both types of acidic cells treated with vemurafenib did not change their proliferation, distribution in cell cycle and level of p-AKT, in contrast to cells grown at standard pH, which showed reduced proliferation, cell cycle arrest and ERK/AKT inhibition. Even after treatment with trametinib (MEK inhibitor) acidic cell features did not change. Then, since both types of acidic cells exhibited high p-p70S6K, i.e. active mTOR signaling, we tested everolimus, an mTOR inhibitor, which was efficient in inducing apoptosis in acidic cells without affecting melanoma cells grown at standard pH. Our results indicate that an acidic microenvironment may cooperate in inducing a BRAF inhibitor resistance in melanoma cells and a combined therapy with everolimus could be used to overcome that resistance. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Cisplatin fails to induce puma mediated apoptosis in mucosal melanomas

    PubMed Central

    Fritsche, Marie Kristin; Metzler, Veronika; Becker, Karen; Plettenberg, Christian; Heiser, Clemens; Hofauer, Benedikt; Knopf, Andreas

    2015-01-01

    Objectives Mucosal melanomas (MM) are aggressive subtypes of common melanomas. It remains unclear whether limitations in their resectability or their distinctive molecular mechanisms are responsible for the aggressive phenotype. Methods In total, 112 patients with cutaneous melanomas (CM) and 27 patients with MM were included. Clinical parameters were analysed using Chi square, Fisher exact and student's t-test. Survival rates were calculated by Kaplan–Meier. Analysis of p53, p21, Mdm2, Hipk2, Gadd45, Puma, Bax, Casp9 and Cdk1 via quantitative PCR and immunohistochemistry (IHC) was performed. TP53 induction after cisplatin treatment was analysed in 10 cell lines (melanocytes, four MM and five CM) using western blot (WB) and qPCR. Results The overall/recurrence-free survival differed significantly between MM (40 months and 30 months) and CM (90 months and 107 months; p < 0.001). IHC and WB confirmed high p53 expression in all melanomas. Hipk2 and Gadd45 showed significantly higher expressions in CM (p < 0.005; p = 0.004). QPCR and WB of wild-type cell lines demonstrated no differences for p53, p21, Mdm2, Bax and Casp9. WB failed to detect Puma in MM, while Cdk1 regulation occurred exclusively in MM. Conclusions The aggressive phenotype of MM did not appear to be due to differential expressions of p53, p21, Mdm2, Bax or Casp9. A non-functional apoptosis in MM may have further clinical implications. PMID:25831048

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

    PubMed

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

    1989-05-01

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

  5. [Study of negative feedback between wild-type BRAF or RAFV600E and Mps1 in melanoma].

    PubMed

    Zhang, Ling; He, Chanting; Bi, Yanghui; Liu, Feng; Cui, Heyang; Wang, Juan; Song, Bin; Shi, Ruyi; Yang, Bin; Wang, Fang; Jia, Zhiwu; Zhao, Zhenxiang; Liu, Jing

    2015-04-01

    To study the effect of Mps1 on BRAFWT/MEK/ERK pathway in the presence of wild type BRAF or BRAFV600E in melanoma. Melanoma cells harboring BRAFWT genotype were transfected either with pBabe-puro-GST-BRAF-WT and/or pBabe-puro-GFP-Mps1-WT or pBabe-puro-GST-BRAFV600E and/or pBabe-puro-GFP-Mps1-WT, followed by Western blot to detect Mps1 and p-ERK expression. The melanoma cells harboring BRAFWT and BRAFV600E genotype were infected with pSUPER-Mps1 retrovirus to knockdown the endogenous Mps1 protein, followed by Western blot to detect Mps1 and p-ERK expression. Meanwhile, melanoma cells harboring BRAFV600E genotype were infected with pBabe-puro-GFP-Mps1 and Western blot was performed to detect Mps1 and p-ERK expression. In melanoma cells harboring BRAFWT genotype and transfected with pBabe-puro-GST-BRAF-WT and pBabe-puro-GFP-Mps1-WT, phospho-ERK levels were notably reduced as compared to either negative control or empty vector. However, cells transfected with pBabe-puro-GST-BRAFV600E and pBabe-puro-GFP-Mps1-WT, phospho-ERK levels did not change significantly compared with either negative control or empty vector. Knockout of Mps1 in BRAF wild-type cell lines led to an increased ERK activity. However, there was no significant change of ERK activity in BRAFV600E cell lines in the absence of Mps1. The expression of p-ERK in BRAFV600E mutant cell lines infected with pBabe-puro-GFP-Mps1-WT did not show any significant difference from either negative control or empty vector. Based on these findings, it suggests that there exists an auto-regulatory negative feedback loop between the Mps1 kinase and BRAFWT/ERK signaling. Oncogenic BRAFV600E abrogates the regulatory negative feedback loop of Mps1 on the MAPK pathway.

  6. Activation of the MAPK pathway is a common event in uveal melanomas although it rarely occurs through mutation of BRAF or RAS.

    PubMed

    Zuidervaart, W; van Nieuwpoort, F; Stark, M; Dijkman, R; Packer, L; Borgstein, A-M; Pavey, S; van der Velden, P; Out, C; Jager, M J; Hayward, N K; Gruis, N A

    2005-06-06

    In contrast to cutaneous melanoma, there is no evidence that BRAF mutations are involved in the activation of the mitogen-activated protein kinase (MAPK) pathway in uveal melanoma, although there is increasing evidence that this pathway is activated frequently in the latter tumours. In this study, we performed mutation analysis of the RAS and BRAF genes in a panel of 11 uveal melanoma cell lines and 19 primary uveal melanoma tumours. In addition, Western blot and immunohistochemical analyses were performed on downstream members of the MAPK pathway in order to assess the contribution of each of these components. No mutations were found in any of the three RAS gene family members and only one cell line carried a BRAF mutation (V599E). Despite this, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK), ERK and ELK were constitutively activated in all samples. These data suggest that activation of the MAPK pathway is commonly involved in the development of uveal melanoma, but occurs through a mechanism different to that of cutaneous melanoma.

  7. Activation of the MAPK pathway is a common event in uveal melanomas although it rarely occurs through mutation of BRAF or RAS

    PubMed Central

    Zuidervaart, W; van Nieuwpoort, F; Stark, M; Dijkman, R; Packer, L; Borgstein, A-M; Pavey, S; van der Velden, P; Out, C; Jager, M J; Hayward, N K; Gruis, N A

    2005-01-01

    In contrast to cutaneous melanoma, there is no evidence that BRAF mutations are involved in the activation of the mitogen-activated protein kinase (MAPK) pathway in uveal melanoma, although there is increasing evidence that this pathway is activated frequently in the latter tumours. In this study, we performed mutation analysis of the RAS and BRAF genes in a panel of 11 uveal melanoma cell lines and 19 primary uveal melanoma tumours. In addition, Western blot and immunohistochemical analyses were performed on downstream members of the MAPK pathway in order to assess the contribution of each of these components. No mutations were found in any of the three RAS gene family members and only one cell line carried a BRAF mutation (V599E). Despite this, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK), ERK and ELK were constitutively activated in all samples. These data suggest that activation of the MAPK pathway is commonly involved in the development of uveal melanoma, but occurs through a mechanism different to that of cutaneous melanoma. PMID:15928660

  8. Cinnamic acid derivatives induce cell cycle arrest in carcinoma cell lines.

    PubMed

    Sova, Matej; Žižak, Željko; Stanković, Jelena A Antic; Prijatelj, Matevž; Turk, Samo; Juranić, Zorica D; Mlinarič-Raščan, Irena; Gobec, Stanislav

    2013-08-01

    Cinnamic acid derivatives can be found in plant material, and they possess a remarkable variety of biological effects. In the present study, we have investigated the cytotoxic effects of representative cinnamic acid esters and amides. The cytotoxicity was determined by MTT test on human cervix adenocarcinoma (HeLa), myelogenous leukemia (K562), malignant melanoma (Fem-x), and estrogen-receptor-positive breast cancer (MCF-7) cells, versus peripheral blood mononuclear cells (PBMCs) without or with the addition of the plant lectin phytohemaglutinin (PHA). The compounds tested showed significant cytotoxicity (IC50s between 42 and 166 µM) and furthermore selectivity of these cytotoxic effects on the malignant cell lines versus the PBMCs was also seen, especially when electron-withdrawing groups, such as a cyano group (compound 5), were present on the aromatic rings of the alcohol or amine parts of the cinnamic acid derivatives. The additional study on cell cycle phase distribution indicated that novel cinnamic acid derivatives inhibit cell growth by induction of cell death. Thus, cinnamic acids derivatives represent important lead compounds for further development of antineoplastic agents.

  9. The Effect of Bornyl cis-4-Hydroxycinnamate on Melanoma Cell Apoptosis Is Associated with Mitochondrial Dysfunction and Endoplasmic Reticulum Stress

    PubMed Central

    Yang, Tzu-Yen; Wu, Yu-Jen; Chang, Chi-I; Wu, Mei-Li

    2018-01-01

    Bornyl cis-4-hydroxycinnamate, an active compound isolated from Piper betle stems, was investigated in terms of its effects on A2058 and A375 melanoma cell proliferation and protein expression in this study. We used flow cytometric analysis to examine the early stages of apoptosis induced by bornyl cis-4-hydroxycinnamate in the two melanoma cell lines and employed comparative proteomic analysis to investigate the effects of this compound on protein expression in A375 cells. Master maps generated by PDQuest software from two-dimensional electrophoresis (2-DE) analysis of A375 cells showed that the expression levels of 35 proteins were significantly altered, with 18 proteins upregulated and 17 downregulated. The proteomics study identified several proteins that are involved in mitochondrial dysfunction and endoplasmic reticulum stress (ER stress), in addition to apoptosis-associated proteins, including prohibitin, hypoxia-upregulated protein 1, stress 70 protein, 78 kDa glucose-regulated protein (GRP78), and protein deglycase DJ-1 (protein DJ-1) in melanoma cells exposed to bornyl cis-4-hydroxycinnamate. The treatment also resulted in a marked decline of the mitochondrial membrane potential, in cytochrome C release into the cytosol, in the activation of Bcl-2-associated X protein (Bax), Bcl-2-associated death promoter protein (Bad), caspase-3, and caspase-9, and in the decreased expression of p-Bad, B-cell lymphoma 2 (Bcl-2), Bcl-xl, and induced myeloid leukemia cell differentiation protein-1 (Mcl-1), indicating that apoptosis induced by bornyl cis-4-hydroxycinnamate was mediated by the mitochondria through the caspase-dependent pathway. Also, salubrinal (an eukaryotic initiation factor 2α inhibitor; eIF2α inhibitor) was able to protect the cells from bornyl cis-4-hydroxycinnamate-induced apoptosis. Bornyl cis-4-hydroxycinnamate-related cell death also implied that the protein kinase R-like endoplasmic reticulum kinase (PERK)–eIF2α–ATF4–CHOP signal pathways

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

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

    Jorgensen, Kjersti; Skrede, Martina; Cruciani, Veronique

    2005-04-01

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

  11. siRNA - Mediated LRP/LR knock-down reduces cellular viability of malignant melanoma cells through the activation of apoptotic caspases.

    PubMed

    Rebelo, Thalia M; Vania, Leila; Ferreira, Eloise; Weiss, Stefan F T

    2018-07-01

    The 37 kDa/67 kDa laminin receptor (LRP/LR) is over-expressed in tumor cells and has been implicated in several tumourigenic processes such as metastasis and telomerase activation, however, more importantly the focus of the present study is on the maintenance of cellular viability and the evasion of apoptosis. The aim of the study was to investigate the role of LRP/LR on the cellular viability of early (A375) and late stage (A375SM) malignant melanoma cells. Flow cytometry and western blot analysis revealed that A375SM cells contain more cell-surface and total LRP/LR levels in comparison to the A375 cells, respectively. In order to determine the effect of LRP/LR on cell viability and apoptosis, LRP was down-regulated via siRNA technology. MTT assays revealed that LRP knock-down led to significant reductions in the viability of A375 and A375SM cells. Confocal microscopy indicated nuclear morphological changes suggestive of apoptotic induction in both cell lines and Annexin-V FITC/PI assays confirmed this observation. Additionally, caspase-3 activity assays revealed that apoptosis was induced in both cell lines after siRNA-mediated down-regulation of LRP. Caspase-8 and -9 activity assays suggested that post LRP knock-down; A375 cells undergo apoptosis solely via the extrinsic pathway, while A375SM cells undergo apoptosis via the intrinsic pathway. siRNAs mediated LRP knock-down might represent a powerful alternative therapeutic strategy for the treatment of malignant melanoma through the induction of apoptosis. Copyright © 2018. Published by Elsevier Inc.

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

    PubMed Central

    Lonsdorf, Anke S.; Krämer, Björn F.; Fahrleitner, Manuela; Schönberger, Tanja; Gnerlich, Stephan; Ring, Sabine; Gehring, Sarah; Schneider, Stefan W.; Kruhlak, Michael J.; Meuth, Sven G.; Nieswandt, Bernhard; Gawaz, Meinrad; Enk, Alexander H.; Langer, Harald F.

    2012-01-01

    A mutual relationship exists between metastasizing tumor cells and components of the coagulation cascade. The exact mechanisms as to how platelets influence blood-borne metastasis, however, remain poorly understood. Here, we used murine B16 melanoma cells to observe functional aspects of how platelets contribute to the process of hematogenous metastasis. We found that platelets interfere with a distinct step of the metastasis cascade, as they promote adhesion of melanoma cells to the endothelium in vitro under shear conditions. Constitutively active platelet receptor GPIIb/IIIa (integrin αIIbβ3) expressed on Chinese hamster ovary cells promoted melanoma cell adhesion in the presence of fibrinogen, whereas blocking antibodies to aνβ3 integrin on melanoma cells or to GPIIb/IIIa significantly reduced melanoma cell adhesion to platelets. Furthermore, using intravital microscopy, we observed functional platelet-melanoma cell interactions, as platelet depletion resulted in significantly reduced melanoma cell adhesion to the injured vascular wall in vivo. Using a mouse model of hematogenous metastasis to the lung, we observed decreased metastasis of B16 melanoma cells to the lung by treatment with a mAb blocking the aν subunit of aνβ3 integrin. This effect was significantly reduced when platelets were depleted in vivo. Thus, the engagement of GPIIb/IIIa with aνβ3 integrin interaction mediates tumor cell-platelet interactions and highlights how this interaction is involved in hematogenous tumor metastasis. PMID:22102277

  13. Advances in the Treatment of Metastatic Melanoma: Adoptive T Cell Therapy

    PubMed Central

    Bernatchez, Chantale; Radvanyi, Laszlo G.; Hwu, Patrick

    2012-01-01

    Metastatic melanoma is notoriously resistant to chemotherapy and radiotherapy regimens. The prospect for newly diagnosed metastatic melanoma patients is grim with a median survival of less than a year. Currently, the only therapies resulting in long term disease free intervals, high dose Interleukin-2 (IL-2) and more recently anti-CTLA-41, work through activation of the immune system. However, with both therapies the response rate is low. Advances in our knowledge of how the immune system interacts with cancer have led to a number of strategies to manipulate anti-tumor immune responses through immunotherapy. This review will focus on one avenue of immunotherapy using the transfer of T cells referred to as “Adoptive Cell Therapy” (ACT), which involves the ex vivo expansion of autologous tumor-specific T cells to large numbers that are ultimately transferred back to the patient to boost anti-tumor immunity. This approach has been shown to be effective in the treatment of virally induced cancers, as well as metastatic melanoma. Recent successes with ACT hold promise and further emphasize the tremendous potential benefit of harnessing the immune system in the fight against cancer. PMID:22484193

  14. Photodynamic therapy mediated by acai oil (Euterpe oleracea Martius) in nanoemulsion: A potential treatment for melanoma.

    PubMed

    Monge-Fuentes, Victoria; Muehlmann, Luis Alexandre; Longo, João Paulo Figueiró; Silva, Jaqueline Rodrigues; Fascineli, Maria Luiza; de Souza, Paulo; Faria, Fernando; Degterev, Igor Anatolievich; Rodriguez, Anselmo; Carneiro, Fabiana Pirani; Lucci, Carolina Madeira; Escobar, Patricia; Amorim, Rivadávio Fernandes Batista; Azevedo, Ricardo Bentes

    2017-01-01

    Melanoma is the most aggressive and lethal form of skin cancer, responsible for >80% of deaths. Standard treatments for late-stage melanoma usually present poor results, leading to life-threatening side effects and low overall survival. Thus, it is necessary to rethink treatment strategies and design new tools for the treatment of this disease. On that ground, we hereby report the use of acai oil in nanoemulsion (NanoA) as a novel photosensitizer for photodynamic therapy (PDT) used to treat melanoma in in vitro and in vivo experimental models. NIH/3T3 normal cells and B16F10 melanoma cell lines were treated with PDT and presented 85% cell death for melanoma cells, while maintaining high viability in normal cells. Flow cytometry indicated that cell death occurred by late apoptosis/necrosis. Tumor bearing C57BL/6 mice treated five times with PDT using acai oil in nanoemulsion showed tumor volume reduction of 82% in comparison to control/tumor group. Necrotic tissue per tumor area reached its highest value in PDT-treated mice, supporting PDT efficacy. Overall, acai oil in nanoemulsion was an effective photosensitizer, representing a promising source of new photosensitizing molecules for PDT treatment of melanoma, a tumor with an inherent tendency to be refractory for this type of therapy. Copyright © 2016. Published by Elsevier B.V.

  15. The combination of MLN2238 (ixazomib) with interferon-alpha results in enhanced cell death in melanoma.

    PubMed

    Suarez-Kelly, Lorena P; Kemper, Gregory M; Duggan, Megan C; Stiff, Andrew; Noel, Tiffany C; Markowitz, Joseph; Luedke, Eric A; Yildiz, Vedat O; Yu, Lianbo; Jaime-Ramirez, Alena Cristina; Karpa, Volodymyr; Zhang, Xiaoli; Carson, William E

    2016-12-06

    The ubiquitin-proteasome signaling pathway is critical for cell cycle regulation and neoplastic growth. Proteasome inhibition can activate apoptotic pathways. Bortezomib, a selective proteasome inhibitor, has anti-melanoma activity. MLN2238 (ixazomib), an oral proteasome inhibitor, has improved pharmacotherapeutic parameters compared to bortezomib. Interferon-alpha (IFN-α), an immune boosting agent, is FDA-approved for treatment of melanoma. In this study in vitro and in vivo evaluation of the antitumor potential of ixazomib and combination treatments with ixazomib and IFN-α were performed. Apoptosis induced by ixazomib was first observed at 12 hours and was maximal at 48 hours with similar levels of cell death compared to bortezomib. IFN-α alone had little effect on cell viability in vitro. However, the combination of ixazomib with IFN-α significantly enhanced ixazomib's ability to induce apoptotic cell death in BRAF V600E mutant and BRAF wild-type human melanoma tumor cells. The combination of ixazomib and IFN-α also enhanced inhibition of cell proliferation in BRAF V600E mutant melanoma tumor cells; however, this was not seen in BRAF wild-type cells. Ixazomib-induced apoptosis was associated with processing of the pro-apoptotic proteins procaspase-3, -7, -8, and -9, and cleavage of poly-ADP-ribose polymerase (PARP). In an in vivo xenograft model of human melanoma, combination treatment with IFN-α-2b and ixazomib demonstrated a significant reduction in tumor volume when compared to vehicle (p = 0.005) and single therapy ixazomib (p = 0.017) and IFN-α-2b (p = 0.036). These pre-clinical results support further evaluation of combination treatment with ixazomib and IFN-α for the treatment of advanced BRAF V600E mutant melanoma.

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

    PubMed Central

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

    2013-01-01

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

  17. Flavonoids in Ginkgo biloba fallen leaves induce apoptosis through modulation of p53 activation in melanoma cells.

    PubMed

    Park, Hye-Jung; Kim, Moon-Moo

    2015-01-01

    The aim of the present study was to examine the apoptotic effect of flavonoids in methanol extracts of Ginkgo biloba fallen leaves (MEGFL) on melanoma cells. Ginkgo biloba is a deciduous castle chaplain and its leaves include various types of flavonoids such as flavonol-O-glycosides. Ginkgo biloba is known to have therapeutic properties against a number of diseases such as cerebrovascular diseases, blood circulation disease and hypertension. In the present study MEGFL exhibited a higher cytotoxic effect on melanoma cells than Ginkgo biloba leaves (MEGL). It was also found that MEGFL induced apoptotic cell death which was characterized by DNA fragmentation. During the cell death process following treatment with MEGFL, the expression of a variety of death-associated proteins including p53, caspase-3, caspase-9, cytochrome c and Bax were analyzed in the cytosol of melanoma cells. MEGFL significantly increased the expression levels of caspase-3, caspase-9 and p53 in a dose-dependent manner. Our results indicate that MEGFL induced apoptotic cell death by increasing the expression of cell death-associated proteins in melanoma cells.

  18. [Telomerase activity in uveal melanomas].

    PubMed

    Rohrbach, J M; Riedinger, C; Wild, M; Partsch, M

    2000-05-01

    The maximum number of cell divisions of a certain cell population is genetically fixed so that aging cells become non-dividing (senescent) at least. This replicative life span, also known as "Hayflick limit", is probably defined by a "critical" length of the telomeres. Telomeres are special DNA-sequences located at the four ends of the chromosomes which are shortened with each cell cycle. Cells of most, but not all malignant tumours have been shown to reactivate the enzyme telomerase so that telomeres can be reconstructed, "Hayflick limit" can be overcome, and unlimited cell division can be established. This study was undertaken to elucidate whether telomerase reactivation is used by uveal melanoma cells. Fresh tumour tissue was removed from 10 untreated uveal melanomas after enucleation. Telomerase activity was determined using a PCR ELISA according to the Telomeric Repeat Amplification Protocol (TRAP). Normal tissue of the skin and the conjunctiva served as control. Telomerase activity was detectable in 90% of the investigated uveal melanomas. All control specimens were telomerase negative. Uveal melanoma growth seems to depend on telomerase reactivation. Thus, telomerase inhibition could offer a new principle for uveal melanoma therapy in the future.

  19. Inhibitory effect of six green tea catechins and caffeine on the growth of four selected human tumor cell lines.

    PubMed

    Valcic, S; Timmermann, B N; Alberts, D S; Wächter, G A; Krutzsch, M; Wymer, J; Guillén, J M

    1996-06-01

    Green tea is an aqueous infusion of dried unfermented leaves of Camellia sinensis (family Theaceae) from which numerous biological activities have been reported including antimutagenic, antibacterial, hypocholesterolemic, antioxidant, antitumor and cancer preventive activities. From the aqueous-alcoholic extract of green tea leaves, six compounds (+)-gallocatechin (GC), (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), (-)-epigallocatechin gallate (EGCG) and caffeine, were isolated and purified. Together with (+)-catechin, these compounds were tested against each of four human tumor cells lines (MCF-7 breast carcinoma, HT-29 colon carcinoma, A-427 lung carcinoma and UACC-375 melanoma). The three most potent green tea components against all four tumor cell lines were EGCG, GC and EGC. EGCG was the most potent of the seven green tea components against three out of the four cell lines (i.e. MCF-7 breast cancer, HT-29 colon cancer and UACC-375 melanoma). On the basis of these extensive in vitro studies, it would be of considerable interest to evaluate all three of these components in comparative preclinical in vivo animal tumor model systems before final decisions are made concerning which of these potential chemopreventive drugs should be taken into broad clinical trials.

  20. Enhanced expression of PD-1 and other activation markers by CD4+ T cells of young but not old patients with metastatic melanoma.

    PubMed

    van den Brom, Rob R H; van der Geest, Kornelis S M; Brouwer, Elisabeth; Hospers, Geke A P; Boots, Annemieke M H

    2018-06-01

    The biological behavior of melanoma is unfavorable in the elderly when compared to young subjects. We hypothesized that differences in T-cell responses might underlie the distinct behavior of melanoma in young and old melanoma patients. Therefore, we investigated the circulating T-cell compartment of 34 patients with metastatic melanoma and 42 controls, which were classified as either young or old. Absolute numbers of CD4+ T cells were decreased in young and old melanoma patients when compared to the age-matched control groups. Percentages of naive and memory CD4+ T cells were not different when comparing old melanoma patients to age-matched controls. Percentages of memory CD4+ T cells tended to be increased in young melanoma patients compared to young controls. Proportions of naive CD4+ T cells were lower in young patients than in age-matched controls, and actually comparable to those in old patients and controls. This was accompanied with increased percentages of memory CD4+ T cells expressing HLA-DR, Ki-67, and PD-1 in young melanoma patients in comparison to the age-matched controls, but not in old patients. Proportions of CD45RA-FOXP3 high memory regulatory T cells were increased in young and old melanoma patients when compared to their age-matched controls, whereas those of CD45RA+FOXP3 low naive regulatory T cells were similar. We observed no clear modulation of the circulating CD8+ T-cell repertoire in melanoma patients. In conclusion, we show that CD4+ T cells of young melanoma patients show signs of activation, whereas these signs are less clear in CD4+ T cells of old patients.