scholarly journals Plasticity of Drug-Naïve and Vemurafenib- or Trametinib-Resistant Melanoma Cells in Execution of Differentiation/Pigmentation Program

2019 ◽  
Vol 2019 ◽  
pp. 1-15 ◽  
Author(s):  
Malgorzata Czyz ◽  
Malgorzata Sztiller-Sikorska ◽  
Anna Gajos-Michniewicz ◽  
Marta Osrodek ◽  
Mariusz L. Hartman
Keyword(s):  
Cell Lines ◽  
Melanoma Cell ◽  
Treated Patient ◽  
Melanoma Cells ◽  
Drug Resistant ◽  
Loss Of Function ◽  
Acquired Drug Resistance ◽  
Drug Naïve ◽  
Melanoma Cell Lines ◽  
Resistant Cells

Melanoma plasticity creates a plethora of opportunities for cancer cells to escape treatment. Thus, therapies must target all cancer cell subpopulations bearing the potential to contribute to disease. The role of the differentiation/pigmentation program in intrinsic and acquired drug resistance is largely uncharacterized. MITF level and expression of MITF-dependent pigmentation-related genes, MLANA, PMEL, TYR, and DCT, in drug-naïve and vemurafenib- or trametinib-treated patient-derived melanoma cell lines and their drug-resistant counterparts were analysed and referred to genomic alterations. Variability in execution of pigmentation/differentiation program was detected in patient-derived melanoma cell lines. Acute treatment with vemurafenib or trametinib enhanced expression of pigmentation-related genes in MITF-Mhigh melanoma cells, partially as the consequence of transcriptional reprograming. During development of resistance, changes in pigmentation program were not unidirectional, but also not universal as expression of different pigmentation-related genes was diversely affected. In selected resistant cell lines, differentiation/pigmentation was promoted and might be considered as one of drug-tolerant phenotypes. In other resistant lines, dedifferentiation was induced. Upon drug withdrawal (“drug holiday”), the dedifferentiation process in resistant cells either was enhanced but reversed by drug reexposure suggesting involvement of epigenetic mechanisms or was irreversible. The irreversible dedifferentiation might be connected with homozygous loss-of-function mutation in MC1R, as MC1RR151C  +/+ variant was found exclusively in drug-naïve MITF-Mlow dedifferentiated cells and drug-resistant cells derived from MITFhigh/MC1RWT cells undergoing irreversible dedifferentiation. MC1RR151C  +/+ variant might be further investigated as a parameter potentially impacting melanoma patient stratification and aiding in treatment decision.

scholarly journals Vemurafenib resistant melanoma cells acquire mesenchymal stem cell-like properties

2019 ◽  
Vol 6 (4) ◽  
pp. 47-57
Author(s):  
A. A. Vartanian ◽  
O. S. Burova ◽  
Kh. S. Vishnyakova ◽  
I. V. Samoylenko ◽  
V. A. Misyurin ◽  
...  
Keyword(s):  
Cell Lines ◽  
Melanoma Cell ◽  
Acquired Resistance ◽  
Melanoma Cells ◽  
Mapk Signaling ◽  
Brafv600e Mutation ◽  
Acquired Drug Resistance ◽  
Melanoma Patients ◽  
Braf Mutant ◽  
Melanoma Cell Lines

Background. Activating mutations in the BRAF gene leads to a constitutive activation of the MAPK signaling. The highly selective BRAFV600E inhibitor, vemurafenib, improves the overall survival of BRAF-mutant melanoma patients. However, despite the excellent results of response rate, the average duration of the response was short and acquired resistance develops in most BRAF mutated melanoma patients within a few months. Objective: to derive melanoma cell lines from surgical species of patients with BRAF mutant melanomas resistant to vemurafenib and to elucidate the mechanisms involved in acquired drug resistance.Materials and methods. Mel Ki and Mel F1702 melanoma cells were obtained from metastases of disseminated melanoma patients with BRAFV600E mutation. 2D tumor cell culture, MTT test, immunicytochemistry, flow cytometry, real-time polimerase chain reaction and osteogenic and adipocytic differentiation were used in the study.Results. We have derived two melanoma cell lines Mel Ki and Mel F1702 from tumor samples of patients with BRAFV600E mutation resistant to vemurafenib. These cells were homogenous and had fibroblastic morphology. The IC50 values for Mel Ki and Mel F1702 were 4.7 and 6.3 μM, respectively. The expression of cancer-testis antigens was not detected in both types of cells suggesting the stemness of Mel Ki and Mel F1702 melanoma cells. The immunophenotypic profile of the vemurafenib resistsant melanoma cells showed the expression of typical mesenchymal stem cells markers such as CD90, CD105 and CD44. In addition, we found that the melanoma cell lines derived from tumor resistant to vemurafenib differentiated into osteoblastand adipocyte-like cells. Conclusion. In this study we are offering an experimental evidence of the phenotypic transition of the vemurafenib-resistant melanoma cells into mesenchymal stem-like cells.

scholarly journals Synergy, Additivity, and Antagonism between Cisplatin and Selected Coumarins in Human Melanoma Cells

2021 ◽  
Vol 22 (2) ◽  
pp. 537
Author(s):  
Paula Wróblewska-Łuczka ◽  
Aneta Grabarska ◽  
Magdalena Florek-Łuszczki ◽  
Zbigniew Plewa ◽  
Jarogniew J. Łuszczki
Keyword(s):  
Cell Lines ◽  
Melanoma Cell ◽  
Melanoma Cells ◽  
Human Melanoma ◽  
Type I ◽  
Antiproliferative Effects ◽  
Isobolographic Analysis ◽  
Natural Substances ◽  
Additive Interactions ◽  
Melanoma Cell Lines

(1) Cisplatin (CDDP) is used in melanoma chemotherapy, but it has many side effects. Hence, the search for natural substances that can reduce the dose of CDDP, and CDDP-related toxicity, is highly desired. Coumarins have many biological properties, including anticancer and antiproliferative effects. (2) An in vitro 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay on two human melanoma cell lines (FM55P and FM55M2) examined the antitumor properties of CDDP and five naturally occurring coumarins (osthole, xanthotoxin, xanthotoxol, isopimpinellin, and imperatorin). The antiproliferative effects produced by combinations of CDDP with the coumarins were assessed using type I isobolographic analysis. (3) The most potent anticancer properties of coumarins were presented by osthole and xanthotoxol. These compounds were characterized by the lowest median inhibitory concentration (IC50) values relative to the FM55P and FM55M2 melanoma cells. Isobolographic analysis showed that for both melanoma cell lines, the combination of CDDP and osthole exerted synergistic and additive interactions, while the combination of CDDP and xanthotoxol exerted additive interactions. Combinations of CDDP with xanthotoxin, isopimpinellin, and imperatorin showed antagonistic and additive interactions in two melanoma cell lines. (4) The combination of CDDP and osthole was characterized by the most desirable synergistic interaction. Isobolographic analysis allows the selection of potential candidates for cancer drugs among natural substances.

Secretion of Cytokines by Human Choroidal Melanoma Cells and Skin Melanoma Cell Lines in vitro

1998 ◽  
Vol 30 (3) ◽  
pp. 189-194 ◽  
Author(s):  
Volker Enzmann ◽  
Frank Faude ◽  
Leon Kohen ◽  
Peter Wiedemann
Keyword(s):  
Cell Lines ◽  
Melanoma Cell ◽  
Choroidal Melanoma ◽  
Melanoma Cells ◽  
Skin Melanoma ◽  
Melanoma Cell Lines

scholarly journals Identification and characterization of a low-affinity granulocyte- macrophage colony-stimulating factor receptor on primary and cultured human melanoma cells

Blood ◽  
1991 ◽  
Vol 78 (3) ◽  
pp. 609-615 ◽  
Author(s):  
GC Baldwin ◽  
DW Golde ◽  
GF Widhopf ◽  
J Economou ◽  
JC Gasson
Keyword(s):  
Cell Lines ◽  
Melanoma Cell ◽  
Melanoma Cells ◽  
Human Melanoma ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor ◽  
Melanoma Cell Lines

Abstract Hematopoietic growth factor receptors are present on cells of normal nonhematopoietic tissues such as endothelium and placenta. We previously demonstrated functional human granulocyte-macrophage colony- stimulating factor (GM-CSF) receptors on small cell carcinoma of the lung cell lines, and others have reported that certain solid tumor cell lines respond to GM-CSF in clonogenic assays. In the current study, we examine human melanoma cell lines and fresh specimens of melanoma to determine whether they have functional GM-CSF receptors. Scatchard analyses of 125I-GM-CSF equilibrium binding to melanoma cell lines showed a mean of 542 +/- 67 sites per cell with a kd of 0.72 +/- 0.14 nmol/L. Cross-linking studies in the melanoma cell line, M14, showed a major GM-CSF receptor species of 84,000 daltons. Under the conditions tested, the M14 cells did not have a proliferative response to GM-CSF in vitro, nor was any induction of primary response genes detected by Northern analysis in response to GM-CSF. Studies to determine internal translocation of the receptor-ligand complex indicated less than 10% of the 125I-GM-CSF internalized was specifically bound to receptors. Primary melanoma cells from five surgical specimens had GM-CSF receptors; Scatchard analysis was performed on one sample, showing 555 sites/cell with a kd of 0.23 nmol/L. These results indicate that human tumor cells may express a low-affinity GM-CSF receptor protein that localizes to the cell surface and binds ligand, but lacks functional components or accessory factors needed to transduce a signal.

Effect of small inhibitors of nuclear export (SINE) on growth inhibition and apoptosis of human melanoma cells.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e13549-e13549
Author(s):  
Gregory B. Lesinski ◽  
Jennifer Yang ◽  
Matthew A Bill ◽  
Yosef Landesman ◽  
Sharon Shacham ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Cell Lines ◽  
Melanoma Cell ◽  
Nuclear Export ◽  
Melanoma Cells ◽  
Human Melanoma ◽  
Human Melanoma Cell ◽  
Nuclear Accumulation ◽  
Dependent Manner ◽  
Melanoma Cell Lines

e13549 Background: Inhibition of nuclear export can promote re-activation of tumor suppressor pathways. CRM1 (chromosomal regional maintenance 1) or XPO1 (exportin 1) is the major protein that mediates nuclear export. We hypothesized that CRM1 mediated nuclear export represents a novel therapeutic target that can be manipulated to inhibit melanoma cell survival. Methods: The growth inhibitory and pro-apoptotic effects of KPT-185, KPT-276 and KPT-330, small molecules selective inhibitor of nuclear export (SINE) were evaluated in human melanoma cell lines using an MTT assay and Annexin V/PI staining, respectively. Fluorescence microscopy and immunoblots were used to assess nuclear accumulation of tumor suppressor proteins. The trans-isomer of KPT-185 and DMSO (vehicle) were used as a negative controls in all assays. The pharmacokinetic (PK) profile of all compounds was evaluated in mice. Results: CRM1 protein was highly expressed in human melanoma cell lines with diverse molecular profiles (i.e., B-Raf, NRAS, p53). KPT-SINE inhibited melanoma cell growth in a concentration-dependent manner and induced apoptosis at nanomolar concentrations. Importantly, there was no evidence that B-Raf V600 mutational status influenced melanoma cell response to these agents. Nuclear accumulation and/or induction of p53, p21, FOXO3a, STAT1 and BAD, and reduction of MCL-1 occurred in melanoma cells at time points prior to apoptosis as shown by increase in cleaved PARP and caspase 3 levels. PK studies were conducted in mice following oral administration of 10 mg/kg, to guide drug selection for our ongoing efficacy studies in murine melanoma models. KPT-185 showed limited bioavailability and systemic exposure, while KPT-276 and KPT-330 showed >50% bioavailability reaching Cmax >5µM. Conclusions: This study represents the first report of CRM1 inhibition in melanoma. These data indicate that the novel SINE compounds can effectively inhibit CRM1-mediated nuclear export and induce apoptosis in melanoma cells. KPT-330 is currently under development as orally bioavailable, small molecule inhibitors for a human clinical trial.

scholarly journals Migration of highly aggressive melanoma cells on hyaluronic acid is associated with functional changes, increased turnover and shedding of CD44 receptors

1996 ◽  
Vol 109 (7) ◽  
pp. 1957-1964 ◽  
Author(s):  
M. Goebeler ◽  
D. Kaufmann ◽  
E.B. Brocker ◽  
C.E. Klein
Keyword(s):  
Hyaluronic Acid ◽  
Cell Lines ◽  
Melanoma Cell ◽  
Malignant Tumors ◽  
Standard Form ◽  
Metastatic Potential ◽  
Melanoma Cells ◽  
Functional Changes ◽  
Local Progression ◽  
Melanoma Cell Lines

Recent evidence indicates that CD44, a multifunctional adhesion receptor involved in cell-cell as well as in cell-matrix interactions, plays an important role in local progression and metastasis of malignant tumors. We have studied a set of human melanoma cell lines differing in their metastatic potential in nude mice as well as in normal melanocytes for changes in CD44 expression and function. All melanocytes and melanoma cell lines tested highly expressed the CD44 standard form (CD44s, 85 kDa) but variants at low levels only. With respect to one of the CD44-associated functions primarily involved in tumor progression we found that two highly metastatic tumor cell lines, MV3 and BLM, showed fivefold higher migration rates towards hyaluronate than melanomas with low metastatic potential and normal melanocytes. Moreover, the highly metastatic cell lines expressed four- to sixfold higher levels of the CD44 epitope involved in hyaluronic acid-binding (monoclonal antibody Hermes-1) than less aggressive melanomas and melanocytes. Hermes-1 efficiently blocked haptotaxis to hyaluronate, supporting the functional relevance of this epitope. In contrast, expression levels of other CD44s epitopes recognized by seven different anti-CD44 monoclonal antibodies were unchanged, suggesting that the migratory behaviour of the cells depends on the formation of the hyaluronate-binding Hermes-1 epitope rather than on the overall CD44s surface expression, which was virtually identical in all melanoma and melanocyte cell lines tested. Differences in the accessibility of the hyaluronate-binding epitope defined by Hermes-1 correlated with the phosphorylation state of CD44s, probably reflecting different activation states of the receptor. Furthermore, immunoprecipitation and pulse/chase studies revealed a three- to fivefold increase in CD44 synthesis in the highly aggressive melanoma cells as compared to the other cell lines and the melanocytes, indicating a reduction of CD44 half-life and up-regulation of turnover. Moreover, highly aggressive melanoma cell lines were found to shed significant amounts of CD44 from the cell surface and to secrete its ligand hyaluronic acid, which may refer to an “autocrine' mechanism mediating melanoma cell motility.

scholarly journals A lipid-free and insulin-supplemented medium supports de novo fatty acid synthesis gene activation in melanoma cells

2018 ◽  
Author(s):  
Su Wu ◽  
Anders M. Näär
Keyword(s):  
Cell Culture ◽  
Fatty Acid ◽  
Cell Lines ◽  
Melanoma Cell ◽  
De Novo ◽  
Melanoma Cells ◽  
Controlled Study ◽  
Gene Promoters ◽  
Medium Condition ◽  
Melanoma Cell Lines

AbstractWhile investigating the role played by de novo lipid (DNL) biosynthesis in cancer cells, we sought a medium condition that would support cell proliferation without providing any serum lipids. Here we report that a defined serum free cell culture medium condition containing insulin, transferrin and selenium (ITS) supports controlled study of transcriptional regulation of de novo fatty acid (DNFA) production and de novo cholesterol synthesis (DNCS) in melanoma cell lines. This lipid-free ITS medium is able to support continuous proliferation of several melanoma cell lines that utilize DNL to support their lipid requirements. We show that the ITS medium stimulates gene transcription in support of both DNFA and DNCS, specifically mediated by SREBP1/2 in melanoma cells. We further found that the ITS medium promoted SREBP1 nuclear localization and occupancy on DNFA gene promoters. Our data show clear utility of this serum and lipid-free medium for melanoma cancer cell culture and lipid-related areas of investigation.

scholarly journals Transcriptional Reprogramming and Constitutive PD-L1 Expression in Melanoma Are Associated with Dedifferentiation and Activation of Interferon and Tumour Necrosis Factor Signalling Pathways

Cancers ◽  
2021 ◽  
Vol 13 (17) ◽  
pp. 4250
Author(s):  
Antonio Ahn ◽  
Euan J. Rodger ◽  
Jyoti Motwani ◽  
Gregory Gimenez ◽  
Peter A. Stockwell ◽  
...  
Keyword(s):  
Gene Expression ◽  
Targeted Therapy ◽  
Tumour Necrosis Factor ◽  
Cell Lines ◽  
Melanoma Cell ◽  
Tumour Necrosis ◽  
Melanoma Cells ◽  
Signalling Pathways ◽  
Necrosis Factor ◽  
Melanoma Cell Lines

Melanoma is the most aggressive type of skin cancer, with increasing incidence worldwide. Advances in targeted therapy and immunotherapy have improved the survival of melanoma patients experiencing recurrent disease, but unfortunately treatment resistance frequently reduces patient survival. Resistance to targeted therapy is associated with transcriptomic changes and has also been shown to be accompanied by increased expression of programmed death ligand 1 (PD-L1), a potent inhibitor of immune response. Intrinsic upregulation of PD-L1 is associated with genome-wide DNA hypomethylation and widespread alterations in gene expression in melanoma cell lines. However, an in-depth analysis of the transcriptomic landscape of melanoma cells with intrinsically upregulated PD-L1 expression is lacking. To determine the transcriptomic landscape of intrinsically upregulated PD-L1 expression in melanoma, we investigated transcriptomes in melanomas with constitutive versus inducible PD-L1 expression (referred to as PD-L1CON and PD-L1IND). RNA-Seq analysis was performed on seven PD-L1CON melanoma cell lines and ten melanoma cell lines with low inducible PD-L1IND expression. We observed that PD-L1CON melanoma cells had a reprogrammed transcriptome with a characteristic pattern of dedifferentiated gene expression, together with active interferon (IFN) and tumour necrosis factor (TNF) signalling pathways. Furthermore, we identified key transcription factors that were also differentially expressed in PD-L1CON versus PD-L1IND melanoma cell lines. Overall, our studies describe transcriptomic reprogramming of melanomas with PD-L1CON expression.

Recombinant arginase as a novel anti-melanoma agent

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 12032-12032 ◽  
Author(s):  
E. C. Hsueh ◽  
S. Knebel ◽  
I. Collier ◽  
M. Kadze ◽  
C. Hsueh ◽  
...  
Keyword(s):  
Real Time ◽  
Cell Lines ◽  
Melanoma Cell ◽  
Large Scale ◽  
Melanoma Cells ◽  
Human Melanoma ◽  
Subtilis Strain ◽  
Argininosuccinate Synthetase ◽  
Melanoma Cell Lines

12032 Background: BCT-100 is a recombinant arginase comprised of 329 amino acid residues. Arginase converts arginine to urea and ornithine. Previous studies suggested that melanoma cells were auxotrophic for arginine due to absence of argininosuccinate synthetase (ASS) expression. Thus, we hypothesized that recombinant arginase, BCT-100, is cytotoxic to human melanoma cells and its cytotoxicity correlates with absence of ASS expression. Methods: BCT-100 pegylated recombinant human arginase was manufactured by large scale fermentation of a recombinant B. subtilis strain LLC101 encoded with a human arginase gene. Following fermentation, the recombinant protein was extracted, purified, pegylated, and ultra-dialyzed. Ten established human melanoma cell lines were used. Cells were grown to 90% confluence, harvested, and plated at 104 cells per well in a 96-well plate and co-cultured with increasing concentrations of pegylated BCT-100 for 72 hours. CellTiter 96 Aqueous Non-radioactive Cell Proliferation Assay (Promega, Madison, WI) was used to measure percent viability, with absorbances measured at 490 nm. Total cellular RNA was isolated from established melanoma cell lines converted to cDNA at a concentration of 5 ng/ul. Quantitative real time polymerase chain reaction was performed on a 7300 Real Time PCR System, using Gene Expression Assays for ASS and GAPDH (Applied Biosystems). 10,000 fold standard curves were generated for all samples using GAPDH expression. Results: All ten cell lines demonstrated decreased viability as concentrations of BCT-100 increased. Average IC50 value was 0.11 IU/ml. Eight of the 10 cells lines have IC50 values < 0.1 IU/ml. Of the 8 cell lines with IC50< 0.1 IU/ml, all of them have low or undetectable ASS expression using quantitative RT-PCR. Of the 2cell lines with IC50 > 0.1 IU/ml, ASS expression was detected in 1 of 2. Conclusions: Arginine depletion with recombinant arginase, BCT-100, was cytotoxic to melanoma cells in vitro. The cytotoxic effect of BCT-100 on melanoma cells correlated with expression of argininosuccinate synthetase. BCT-100 is a promising novel agent for treatment of melanoma. Further in vivo experiment with BCT-100 is ongoing. [Table: see text]

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