scholarly journals Altered Expression of Shorter p53 Family Isoforms Can Impact Melanoma Aggressiveness

Cancers ◽  
2021 ◽  
Vol 13 (20) ◽  
pp. 5231
Author(s):  
Ana Tadijan ◽  
Francesca Precazzini ◽  
Nikolina Hanžić ◽  
Martina Radić ◽  
Nicolò Gavioli ◽  
...  
Keyword(s):  
Cell Lines ◽  
Braf Inhibitor ◽  
Tp53 Gene ◽  
Melanoma Cells ◽  
P53 Family ◽  
Altered Expression ◽  
Dna Damaging Agents ◽  
P53 Isoforms ◽  
The Guardian

Cutaneous melanoma is the most aggressive form of skin cancer. Despite the significant advances in the management of melanoma in recent decades, it still represents a challenge for clinicians. The TP53 gene, the guardian of the genome, which is altered in more than 50% of human cancers, is rarely mutated in melanoma. More recently, researchers started to appreciate the importance of shorter p53 isoforms as potential modifiers of the p53-dependent responses. We analyzed the expression of p53 and p73 isoforms both at the RNA and protein level in a panel of melanoma-derived cell lines with different TP53 and BRAF status, in normal conditions or upon treatment with common anti-cancer DNA damaging agents or targeted therapy. Using lentiviral vectors, we also generated stable clones of H1299 p53 null cells over-expressing the less characterized isoforms Δ160p53α, Δ160p53β, and Δ160p53γ. Further, we obtained two melanoma-derived cell lines resistant to BRAF inhibitor vemurafenib. We observed that melanoma cell lines expressed a wide array of p53 and p73 isoforms, with Δ160p53α as the most variable one. We demonstrated for the first time that Δ160p53α, and to a lesser extent Δ160p53β, can be recruited on chromatin, and that Δ160p53γ can localize in perinuclear foci; moreover, all Δ160p53 isoforms can stimulate proliferation and in vitro migration. Lastly, vemurafenib-resistant melanoma cells showed an altered expression of p53 and p73 isoforms, namely an increased expression of potentially pro-oncogenic Δ40p53β and a decrease in tumor-suppressive TAp73β. We therefore propose that p53 family isoforms can play a role in melanoma cells’ aggressiveness.

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 CD133 Is Associated with Increased Melanoma Cell Survival after Multikinase Inhibition

2019 ◽  
Vol 2019 ◽  
pp. 1-19 ◽  
Author(s):  
Cynthia M. Simbulan-Rosenthal ◽  
Anirudh Gaur ◽  
Hengbo Zhou ◽  
Maryam AbdusSamad ◽  
Qing Qin ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Kinase Inhibitors ◽  
Braf Inhibitor ◽  
Mek Inhibitor ◽  
Melanoma Cells ◽  
Stem Cell Marker ◽  
High Dose ◽  
Recurrence Rates ◽  
Mapk Inhibitors

FDA-approved kinase inhibitors are now used for melanoma, including combinations of the MEK inhibitor trametinib, and BRAF inhibitor dabrafenib for BRAFV600 mutations. NRAS-mutated cell lines are also sensitive to MEK inhibitionin vitro, and NRAS-mutated tumors have also shown partial response to MEK inhibitors. However, melanoma still has high recurrence rates due to subpopulations, sometimes described as “melanoma initiating cells,” resistant to treatment. Since CD133 is a putative cancer stem cell marker for different cancers, associated with decreased survival, we examined resistance of patient-derived CD133(+) and CD133(-) melanoma cells to MAPK inhibitors. Human melanoma cells were exposed to increasing concentrations of trametinib and/or dabrafenib, either before or after separation into CD133(+) and CD133(-) subpopulations. In parental CD133-mixed lines, the percentages of CD133(+) cells increased significantly (p<0.05) after high-dose drug treatment. Presorted CD133(+) cells also exhibited significantly greater (p<0.05) IC50s for single and combination MAPKI treatment. siRNA knockdown revealed a causal relationship between CD133 and drug resistance. Microarray and qRT-PCR analyses revealed that ten of 18 ABC transporter genes were significantly (P<0.05) upregulated in the CD133(+) subpopulation, while inhibition of ABC activity increased sensitivity, suggesting a mechanism for increased drug resistance of CD133(+) cells.

scholarly journals Rho-mediated gene transcription promotes BRAF inhibitor resistance in de-differentiated melanoma cells

2018 ◽  
Author(s):  
SA Misek ◽  
KM Appleton ◽  
TS Dexheimer ◽  
EM Lisabeth ◽  
RS Lo ◽  
...  
Keyword(s):  
Cell Lines ◽  
Gene Transcription ◽  
Melanoma Cell ◽  
Cutaneous Melanoma ◽  
Acquired Resistance ◽  
Rho Kinase ◽  
Braf Inhibitor ◽  
Resistant Cell ◽  
Melanoma Cell Lines

AbstractOver half of cutaneous melanoma tumors have BRAFV600E/Kmutations. Acquired resistance to BRAF inhibitors (BRAFi) remains a major hurdle in attaining durable therapeutic responses. In this study we demonstrate that approximately 50-60% of melanoma cell lines with vemurafenib resistance acquiredin vitroshow activation of RhoA family GTPases. In BRAFi-resistant melanoma cell lines and tumors, activation of RhoA is correlated with decreased expression of melanocyte lineage genes. Using a machine learning approach, we built gene expression-based models to predict drug sensitivity for 265 common anti-cancer compounds. We then projected these signatures onto the collection of TCGA cutaneous melanoma and found that poorly differentiated tumors were predicted to have increased sensitivity to multiple Rho kinase (ROCK) inhibitors. Two transcriptional effectors downstream of Rho, MRTF and YAP1, are activated in the RhoHighBRAFi-resistant cell lines, and resistant cells are more sensitive to inhibition of these transcriptional mechanisms. Taken together, these results support the concept of targeting Rho-regulated gene transcription pathways as a promising therapy approach to restore sensitivity to BRAFi-resistant tumors or as a combination therapy to prevent the onset of drug resistance.

scholarly journals Gene Signatures Induced by Ionizing Radiation as Prognostic Tools in an In Vitro Experimental Breast Cancer Model

Cancers ◽  
2021 ◽  
Vol 13 (18) ◽  
pp. 4571
Author(s):  
Gloria M. Calaf ◽  
Leodan A. Crispin ◽  
Debasish Roy ◽  
Francisco Aguayo ◽  
Juan P. Muñoz ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Progression ◽  
Combined Treatment ◽  
Cancer Model ◽  
Altered Expression ◽  
Breast Cancer Model

This study aimed to analyze the expression of genes involved in radiation, using an Affymetrix system with an in vitro experimental breast cancer model developed by the combined treatment of low doses of high linear energy transfer (LET) radiation α particle radiation and estrogen yielding different stages in a malignantly transformed breast cancer cell model called Alpha model. Altered expression of different molecules was detected in the non-tumorigenic Alpha3, a malignant cell line transformed only by radiation and originally derived from the parental MCF-10F human cell line; that was compared with the Alpha 5 cell line, another cell line exposed to radiation and subsequently grown in the presence 17β-estradiol. This Alpha5, a tumorigenic cell line, originated the Tumor2 cell line. It can be summarized that the Alpha 3 cell line was characterized by greater gene expression of ATM and IL7R than control, Alpha5, and Tumor2 cell lines, it presented higher selenoprotein gene expression than control and Tumor2; epsin 3 gene expression was higher than control; stefin A gene expression was higher than Alpha5; and metallothionein was higher than control and Tumor2 cell line. Therefore, radiation, independently of estrogen, induced increased ATM, IL7R, selenoprotein, GABA receptor, epsin, stefin, and metallothioneins gene expression in comparison with the control. Results showed important findings of genes involved in cancers of the breast, lung, nervous system, and others. Most genes analyzed in these studies can be used for new prognostic tools and future therapies since they affect cancer progression and metastasis. Most of all, it was revealed that in the Alpha model, a breast cancer model developed by the authors, the cell line transformed only by radiation, independently of estrogen, was characterized by greater gene expression than other cell lines. Understanding the effect of radiotherapy in different cells will help us improve the clinical outcome of radiotherapies. Thus, gene signature has been demonstrated to be specific to tumor types, hence cell-dependency must be considered in future treatment planning. Molecular and clinical features affect the results of radiotherapy. Thus, using gene technology and molecular information is possible to improve therapies and reduction of side effects while providing new insights into breast cancer-related fields.

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]

scholarly journals In Cellulo Evaluation of the Therapeutic Potential of NHC Platinum Compounds in Metastatic Cutaneous Melanoma

2020 ◽  
Vol 21 (21) ◽  
pp. 7826
Author(s):  
Elsa Charignon ◽  
Mathilde Bouché ◽  
Caroline Clave-Darcissac ◽  
Georges Dahm ◽  
Gabriel Ichim ◽  
...  
Keyword(s):  
Metastatic Melanoma ◽  
Cell Lines ◽  
Cutaneous Melanoma ◽  
Therapeutic Potential ◽  
Braf Inhibitor ◽  
Melanoma Cells ◽  
Resistant Cell ◽  
Dna Double Strand Breaks ◽  
Pyridine Ligand ◽  
Systemic Treatments

We describe here the evaluation of the cytotoxic efficacy of two platinum (II) complexes bearing an N-heterocyclic carbene (NHC) ligand, a pyridine ligand and bromide or iodide ligands on a panel of human metastatic cutaneous melanoma cell lines representing different genetic subsets including BRAF-inhibitor-resistant cell lines, namely A375, SK-MEL-28, MeWo, HMCB, A375-R, SK-MEL-5-R and 501MEL-R. Cisplatin and dacarbazine were also studied for comparison purposes. Remarkably, the iodine-labelled Pt-NHC complex strongly inhibited proliferation of all tested melanoma cells after 1-h exposure, likely due to its rapid uptake by melanoma cells. The mechanism of this inhibitory activity involves the formation of DNA double-strand breaks and apoptosis. Considering the intrinsic chemoresistance of metastatic melanoma cells of current systemic treatments, these findings are promising and could give research opportunities in the future to improve the prognosis of patients suffering from unresectable metastatic melanoma that are not eligible or that do not respond to the most effective drugs available to date, namely BRAF inhibitors and the anti-PD-1 monoclonal antibody (mAb).

scholarly journals Nuclear Localization of BRAFV600E Is Associated with HMOX-1 Upregulation and Aggressive Behavior of Melanoma Cells

Cancers ◽  
2022 ◽  
Vol 14 (2) ◽  
pp. 311
Author(s):  
Mourad Zerfaoui ◽  
Eman Toraih ◽  
Emmanuelle Ruiz ◽  
Youssef Errami ◽  
Abdallah S. Attia ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Nuclear Localization ◽  
Melanoma Cell ◽  
Braf Inhibitor ◽  
Melanoma Cells ◽  
Heme Oxygenase 1 ◽  
Biological Targets ◽  
Melanoma Cell Proliferation

Background: Previously, we have demonstrated that nuclear BRAFV600E is associated with melanoma aggressiveness and vemurafenib resistance. However, the underlying mechanisms of how nuclear localization of BRAFV600E promotes cell aggressiveness have not yet been investigated. Despite therapeutic advancements targeting cutaneous melanoma, unknown cellular processes prevent effective treatment for this malignancy, prompting an urgent need to identify new biological targets. This study aims to explore the association of inducible heme oxygenase 1 (HMOX-1) with nuclear BRAFV600E in promoting melanoma aggressiveness. Methods: Proteomics analysis was performed to identify the interacting partner(s) of nuclear BRAFV600E. Immunohistochemistry was applied to evaluate the levels of HMOX-1 and nuclear BRAFV600E expression in melanoma and adjacent healthy tissues. Immunofluorescence assessed the nuclear localization of BRAFV600E in vemurafenib-resistant A375R melanoma cells. Further study of HMOX-1 knockdown or BRAFV600E overexpression in melanoma cells suggested a role for HMOX-1 in the regulation of cell proliferation in vivo and in vitro. Finally, Western blot analysis was performed to confirm the pathway by which HMOX-1 mediates Akt signaling. Results: Proteomics results showed that HMOX-1 protein expression was 10-fold higher in resistant A375R cells compared to parental counterpart cells. In vitro and in vivo results illustrate that nuclear BRAFV600E promotes HMOX-1 overexpression, whereas HMOX-1 reduction represses melanoma cell proliferation and tumor growth. Mechanistic studies revealed that HMOX-1 was associated with nuclear BRAFV600E localization, thus promoting melanoma proliferation via a persistent activation of the AKT pathway. Conclusions: Our results highlight a previously unknown mechanism in which the nuclear BRAFV600E/HMOX-1/AKT axis plays an essential role in melanoma cell proliferation. Targeting HMOX-1 could be a novel method for treating melanoma patients who develop BRAF inhibitor resistance.

scholarly journals Functional Implication of Netrin Expression in Malignant Melanoma

2009 ◽  
Vol 31 (6) ◽  
pp. 415-422
Author(s):  
Simone Kaufmann ◽  
Silke Kuphal ◽  
Thomas Schubert ◽  
Anja K. Bosserhoff
Keyword(s):  
Malignant Melanoma ◽  
Melanoma Cells ◽  
Transcriptional Level ◽  
Altered Expression ◽  
Invasion And Migration ◽  
Expression Of Genes ◽  
Functional Relevance ◽  
And Migration

Background: Malignant melanoma cells are known to have altered expression of genes supporting proliferation and invasion, however, the expression of molecules of the Netrin family of repellent factors has not been analyzed in melanomas until now.Results: Here, we show that Netrin-1 expression is strongly induced in melanoma cells compared to melanocytes in vivo and in vitro controlled at the transcriptional level via ETS-1. In addition, the expression of the netrin receptor UNC5B was induced and that of UNC5C was reduced in the tumor cells. In order to determine the functional relevance of Netrin-1 expression in malignant melanoma, Netrin expression in melanoma cells was reduced by siRNA attempts and primary human melanocytes were treated with recombinant Netrin-1. The cells showed no changes in proliferation or apoptosis, however, a strong reduction of migratory properties was observed in the melanoma cells after reduction of Netrin expression whereas melanocyte migration was strongly induced by treatment with Netrin.Conclusions: Our study suggests that Netrin-1 promotes melanoma cell invasion and migration and therefore has an important role in the progression of malignant melanoma.

scholarly journals Electrochemotherapy with bleomycin is effective in BRAF mutated melanoma cells and interacts with BRAF inhibitors

2016 ◽  
Vol 50 (3) ◽  
pp. 274-279 ◽  
Author(s):  
Tanja Dolinsek ◽  
Lara Prosen ◽  
Maja Cemazar ◽  
Tjasa Potocnik ◽  
Gregor Sersa
Keyword(s):  
Clonogenic Assay ◽  
Braf Inhibitor ◽  
Melanoma Cells ◽  
Braf V600e ◽  
Concomitant Treatment ◽  
Braf Inhibitors ◽  
Mutation Status ◽  
Mutational Status ◽  
Melanoma Patients

Abstract Background The aim of the study was to explore the effectiveness of electrochemotherapy (ECT) during the treatment of melanoma patients with BRAF inhibitors. Its effectiveness was tested on BRAF mutated and non-mutated melanoma cells in vitro and in combination with BRAF inhibitors. Materials and methods ECT with bleomycin was performed on two human melanoma cell lines, with (SK-MEL-28) or without (CHL-1) BRAF V600E mutation. Cell survival was determined using clonogenic assay to determine the effectiveness of ECT in melanoma cells of different mutation status. Furthermore, the effectiveness of ECT in concomitant treatment with BRAF inhibitor vemurafenib was also determined in BRAF mutated cells SK-MEL-28 with clonogenic assay. Results The survival of BRAF V600E mutated melanoma cells was even lower than non-mutated cells, indicating that ECT is effective regardless of the mutational status of melanoma cells. Furthermore, the synergistic interaction between vemurafenib and ECT with bleomycin was demonstrated in the BRAF V600E mutated melanoma cells. Conclusions The effectiveness of ECT in BRAF mutated melanoma cells as well as potentiation of its effectiveness during the treatment with vemurafenib in vitro implies on clinical applicability of ECT in melanoma patients with BRAF mutation and/or during the treatment with BRAF inhibitors.

scholarly journals Cell Proliferation Is Strongly Associated with the Treatment Conditions of an ER Stress Inducer New Anti-Melanoma Drug in Melanoma Cell Lines

Biomedicines ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 96
Author(s):  
István Szász ◽  
Viktória Koroknai ◽  
Vikas Patel ◽  
Tibor Hajdú ◽  
Tímea Kiss ◽  
...  
Keyword(s):  
Er Stress ◽  
Cell Lines ◽  
Melanoma Cell ◽  
Melanoma Cells ◽  
Culture Conditions ◽  
Driver Mutations ◽  
Published Data ◽  
Starvation Conditions

HA15 is a new anti-melanoma drug that triggers endoplasmic reticulum (ER) stress and causes deleterious effects on melanoma cell viability due to autophagy and apoptosis, regardless of driver mutations or drug resistance. In this study, we investigated the effect of HA15 on the viability/proliferation of BRAFV600E-mutant melanoma cells using different culture conditions. In contrast to the published data, we did not detect significant melanoma cell death under normal culture conditions using HA15 treatment. Indeed, only cells that were cultured under long-term starvation conditions were sensitive to the drug. Quantitative measurements of ER stress and autophagy markers showed that the compound HA15 does not trigger stress alone but synergistically enhances ER stress under starvation conditions. Importantly, we observed that the viability of normal melanocytes decreased significantly with treatment, even at low HA15 concentrations. Finally yet importantly, we were able to generate HA15-resistant cell lines, which failed by Cerezo et al. In summary, HA15 only influences the viability of cells that are starved for several hours before and during treatment. However, this in vitro setting is far from the in vivo conditions. In addition, our data clearly show that melanoma cells can acquire HA15 resistance. Further studies are needed to prove that HA15 is an effective anti-cancer agent.

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