scholarly journals The Benefit of Reactivating p53 under MAPK Inhibition on the Efficacy of Radiotherapy in Melanoma

Cancers ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 1093 ◽  
Author(s):  
Mohammad Krayem ◽  
Malak Sabbah ◽  
Ahmad Najem ◽  
An Wouters ◽  
Filip Lardon ◽  
...  
Keyword(s):  
Mapk Pathway ◽  
Primary Melanoma ◽  
Melanoma Cells ◽  
Tumor Growth Inhibition ◽  
Triple Combination ◽  
Complete Excision ◽  
Triple Combination Therapy ◽  
P53 Expression ◽  
P53 Reactivation

Radiotherapy (RT) in patients with melanoma historically showed suboptimal results, because the disease is often radioresistant due to various mechanisms such as scavenging free radicals by thiols, pigmentary machinery, or enhanced DNA repair. However, radiotherapy has been utilized as adjuvant therapy after the complete excision of primary melanoma and lymph nodes to reduce the rate of nodal recurrences in high-risk patients. The resistance of melanoma cells to radiotherapy may also be in relation with the constitutive activation of the MAPK pathway and/or with the inactivation of p53 observed in about 90% of melanomas. In this study, we aimed to assess the potential benefit of adding RT to BRAF-mutated melanoma cells under a combined p53 reactivation and MAPK inhibition in vitro and in a preclinical animal model. We found that the combination of BRAF inhibition (vemurafenib, which completely shuts down the MAPK pathway), together with p53 reactivation (PRIMA-1Met) significantly enhanced the radiosensitivity of BRAF-mutant melanoma cells. This was accompanied by an increase in both p53 expression and activity. Of note, we found that radiation alone markedly promoted both ERK and AKT phosphorylation, thus contributing to radioresistance. The combination of vemurafenib and PRIMA-1Met caused the inactivation of both MAPK kinase and PI3K/AKT pathways. Furthermore, when combined with radiotherapy, it was able to significantly enhance melanoma cell radiosensitivity. Interestingly, in nude mice bearing melanoma xenografts, the latter triple combination had not only a synergistic effect on tumor growth inhibition, but also a potent control on tumor regrowth in all animals after finishing the triple combination therapy. RT alone had only a weak effect. In conclusion, we provide a basis for a strategy that may overcome the radioresistance of BRAF-mutated melanoma cells to radiotherapy. Whether this will translate into a rational to use radiotherapy in the curative setting in BRAF-mutated melanoma patients deserves consideration.

scholarly journals Celecoxib as a Valuable Adjuvant in Cutaneous Melanoma Treated with Trametinib

2021 ◽  
Vol 22 (9) ◽  
pp. 4387
Author(s):  
Diana Valentina Tudor ◽  
Ioana Bâldea ◽  
Diana Elena Olteanu ◽  
Eva Fischer-Fodor ◽  
Virag Piroska ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Protein Kinase ◽  
Mapk Pathway ◽  
Human Fibroblasts ◽  
In Vitro Study ◽  
Melanoma Cells ◽  
Mitogen Activated Protein Kinase ◽  
Culture Cells ◽  
Nm Range

Background: Melanoma patients stop responding to targeted therapies mainly due to mitogen activated protein kinase (MAPK) pathway re-activation, phosphoinositide 3 kinase/the mechanistic target of rapamycin (PI3K/mTOR) pathway activation or stromal cell influence. The future of melanoma treatment lies in combinational approaches. To address this, our in vitro study evaluated if lower concentrations of Celecoxib (IC50 in nM range) could still preserve the chemopreventive effect on melanoma cells treated with trametinib. Materials and Methods: All experiments were conducted on SK-MEL-28 human melanoma cells and BJ human fibroblasts, used as co-culture. Co-culture cells were subjected to a celecoxib and trametinib drug combination for 72 h. We focused on the evaluation of cell death mechanisms, melanogenesis, angiogenesis, inflammation and resistance pathways. Results: Low-dose celecoxib significantly enhanced the melanoma response to trametinib. The therapeutic combination reduced nuclear transcription factor (NF)–kB (p < 0.0001) and caspase-8/caspase-3 activation (p < 0.0001), inhibited microphthalmia transcription factor (MITF) and tyrosinase (p < 0.05) expression and strongly down-regulated the phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) signaling pathway more significantly than the control or trametinib group (p < 0.0001). Conclusion: Low concentrations of celecoxib (IC50 in nM range) sufficed to exert antineoplastic capabilities and enhanced the therapeutic response of metastatic melanoma treated with trametinib.

scholarly journals Targeting Rad51 as a strategy for the treatment of melanoma cells resistant to MAPK pathway inhibition

2020 ◽  
Vol 11 (7) ◽  
Author(s):  
Elena Makino ◽  
Lisa Marie Fröhlich ◽  
Tobias Sinnberg ◽  
Corinna Kosnopfel ◽  
Birgit Sauer ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Metastatic Melanoma ◽  
Mapk Pathway ◽  
Critical Role ◽  
Melanoma Cells ◽  
Mapk Signaling ◽  
Transcriptional Level ◽  
Mapk Inhibitor

Abstract Rad51 is an essential factor of the homologous recombination DNA repair pathway and therefore plays an important role in maintaining genomic stability. We show that RAD51 and other homologous recombination repair genes are overexpressed in metastatic melanoma cell lines and in melanoma patient samples, which correlates with reduced survival of melanoma patients. In addition, Rad51 expression in melanoma cells was regulated on a transcriptional level by the MAPK signaling pathway with Elk1 as the main downstream transcriptional effector. Most strikingly, melanoma cells which developed resistance towards MAPK inhibitors could be efficiently targeted by Rad51 inhibitors similar to their sensitive counterparts, leading to DNA damage, G2/M arrest and apoptosis. Furthermore, the treatment of MAPK inhibitor resistant cells with Rad51 inhibitors enhances the susceptibility of these cells for MAPK inhibitor treatment in vitro and in vivo. These data indicate that Rad51 plays a critical role in the survival of metastatic melanoma cells and is a promising target for the therapy of melanoma irrespective of its MAPK inhibitor resistance status.

scholarly journals From Proteomic Mapping to Invasion-Metastasis-Cascade Systemic Biomarkering and Targeted Drugging of Mutant BRAF-Dependent Human Cutaneous Melanomagenesis

Cancers ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 2024
Author(s):  
Aikaterini F. Giannopoulou ◽  
Athanassios D. Velentzas ◽  
Athanasios K. Anagnostopoulos ◽  
Adamantia Agalou ◽  
Nikos C. Papandreou ◽  
...  
Keyword(s):  
Metastatic Melanoma ◽  
Epithelial To Mesenchymal Transition ◽  
Hypoxia Inducible Factor ◽  
Primary Melanoma ◽  
Melanoma Cells ◽  
Mesenchymal Transition ◽  
Mesenchymal To Epithelial Transition ◽  
Liquid Chromatography Tandem Mass ◽  
Novel Biomarkers

Melanoma is classified among the most notoriously aggressive human cancers. Despite the recent progress, due to its propensity for metastasis and resistance to therapy, novel biomarkers and oncogenic molecular drivers need to be promptly identified for metastatic melanoma. Hence, by employing nano liquid chromatography-tandem mass spectrometry deep proteomics technology, advanced bioinformatics algorithms, immunofluorescence, western blotting, wound healing protocols, molecular modeling programs, and MTT assays, we comparatively examined the respective proteomic contents of WM115 primary (n = 3955 proteins) and WM266-4 metastatic (n = 6681 proteins) melanoma cells. It proved that WM115 and WM266-4 cells have engaged hybrid epithelial-to-mesenchymal transition/mesenchymal-to-epithelial transition states, with TGF-β controlling their motility in vitro. They are characterized by different signatures of SOX-dependent neural crest-like stemness and distinct architectures of the cytoskeleton network. Multiple signaling pathways have already been activated from the primary melanoma stage, whereas HIF1α, the major hypoxia-inducible factor, can be exclusively observed in metastatic melanoma cells. Invasion-metastasis cascade-specific sub-routines of activated Caspase-3-triggered apoptosis and LC3B-II-dependent constitutive autophagy were also unveiled. Importantly, WM115 and WM266-4 cells exhibited diverse drug response profiles, with epirubicin holding considerable promise as a beneficial drug for metastatic melanoma clinical management. It is the proteome navigation that enables systemic biomarkering and targeted drugging to open new therapeutic windows for advanced disease.

scholarly journals Epigenetic control of melanoma cell invasiveness by the stem cell factor SALL4

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Johanna Diener ◽  
Arianna Baggiolini ◽  
Mattias Pernebrink ◽  
Damian Dalcher ◽  
Luigi Lerra ◽  
...  
Keyword(s):  
Stem Cell ◽  
Transcriptional Profiling ◽  
Embryonic Stem ◽  
Primary Melanoma ◽  
Melanoma Cells ◽  
Tumor Formation ◽  
In Vitro Assays ◽  
Melanoma Model ◽  
Phenotype Switch

AbstractMelanoma cells rely on developmental programs during tumor initiation and progression. Here we show that the embryonic stem cell (ESC) factor Sall4 is re-expressed in the Tyr::NrasQ61K; Cdkn2a−/− melanoma model and that its expression is necessary for primary melanoma formation. Surprisingly, while Sall4 loss prevents tumor formation, it promotes micrometastases to distant organs in this melanoma-prone mouse model. Transcriptional profiling and in vitro assays using human melanoma cells demonstrate that SALL4 loss induces a phenotype switch and the acquisition of an invasive phenotype. We show that SALL4 negatively regulates invasiveness through interaction with the histone deacetylase (HDAC) 2 and direct co-binding to a set of invasiveness genes. Consequently, SALL4 knock down, as well as HDAC inhibition, promote the expression of an invasive signature, while inhibition of histone acetylation partially reverts the invasiveness program induced by SALL4 loss. Thus, SALL4 appears to regulate phenotype switching in melanoma through an HDAC2-mediated mechanism.

scholarly journals GENE SILENCING

2018 ◽  
Vol 25 (12) ◽  
pp. 1954-1960
Author(s):  
Farah Deeba Khan ◽  
Ghazala Irshad ◽  
Samra Hafiz
Keyword(s):  
Cell Proliferation ◽  
Hela Cells ◽  
Mapk Pathway ◽  
In Vitro Study ◽  
Small Gtpase ◽  
Molecular Medicine ◽  
Tumor Growth Inhibition ◽  
Before And After

Objectives: Cancer, the most complex group of genetic disorders results due to over expression or mutation of oncogenes/molecules involved in cell signaling pathways. KRAS is an oncogene that encodes a small GTPase protein with two isoforms KRasA & KRasB and is involved in the regulation of cell division. KRas is frequently found mutated in lung, pancreas, colorectal and many other cancers. Various studies have found that KRasB promotes cell proliferation and inhibits apoptosis whereas KRasA has negligible role in cell proliferation or rather is involved in apoptosis at times. Several experiments have shown tumor growth inhibition by silencing KRas in various tumor models having a differential allelic expression.The goal of our study was to determine the possible differential role of KRas A and B on MAPK Pathway. To examine the disparity in role of various isoforms of KRas on apoptosis, we evaluated the expression of these isoforms through different modalities in HeLa cells before and after silencing KRas through RNA interference. Study Design: In vitro study for isolation of protein molecules (Proteomics) and to study various genes (Genomics) through Polymerase chain reaction. Study Duration: December, 2011-September, 2014. Setting: Center for Research in Molecular Medicine, University of Lahore. Material & Methods: In present study, we studied the expression level and behavior of many sets of molecules such as KRasA, KrasB, Bad, Bcl2, BclxL and Mcl-1 through gene quantitation by Real Time PCR. We also analyzed the protein expression through Western blot immune-precipitation. All the tests were done before and after 48-hours of silencing of HeLa cells with shRNA designed for KRas. Results: We successfully downregulated KRasB (80%) but found upregulation of KRasA with continued cell proliferation. We also found overexpression of antiapoptotic genes, BclxL and Mcl1 and downregulation of proapoptotic molecule-Bad. Differences were considered significant at p< 0.01. Values were expressed as mean ± SEM from six separate experiments. Conclusion: We were able to show that in the absence of one proliferative gene, another sister gene upregulates and takes over the role of uncontrolled cell proliferation. This usually leads to failure of most cancer controltherapies. 

In vitro and in vivo tumor growth inhibition by a p16-mimicking peptide in p16INK4A-defective, pRb-positive human melanoma cells

2004 ◽  
Vol 202 (3) ◽  
pp. 922-928 ◽  
Author(s):  
Douglas M. Noonan ◽  
Anna Severino ◽  
Monica Morini ◽  
Alessandra Tritarelli ◽  
Lucrezia Manente ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Growth Inhibition ◽  
Melanoma Cells ◽  
Human Melanoma ◽  
Tumor Growth Inhibition ◽  
Human Melanoma Cells

scholarly journals Triple Combination Nitazoxanide, Ribavirin, and Hydroxychloroquine results in the multiplicative reduction of in vitro SARS-CoV-2 viral replication

2020 ◽  
Author(s):  
Elena Lian ◽  
Carley McAlister ◽  
Gabriela Ramirez ◽  
David N. Chernoff ◽  
Gregory Went ◽  
...  
Keyword(s):  
Viral Replication ◽  
Low Cost ◽  
Virus Yield ◽  
Triple Combination ◽  
Triple Combination Therapy ◽  
Time To Peak ◽  
Log Reduction ◽  
Additional Cell ◽  
Approved Drugs

AbstractBackgroundAn immediate unmet medical need exists to test and develop existing approved drugs against SARS-COV-2. Despite many efforts, very little progress has been made regarding finding low-cost oral medicines that can be made widely available worldwide to address the global pandemic.MethodsWe sought to examine if a triple combination of nitazoxanide (using its active metabolite tizoxanide), ribavirin, and hydroxychloroquine would lead to a multiplicative effects on viral replication of SARS-COV-2 resulting in a significant reduction of virus yield using VERO E6 cells as a model of viral replication.ResultsVirus yield measured in PFU/ml was ~ 2 logs lower with triple combination versus either drug alone, resulting in the prolongation of time to peak cytopathic effects (CPE). The time to produce 50% CPE increased from 2.8 days for viral controls versus 5.3 days for triple combination therapy. Finally, for each 1-log reduction in virus yield 24 hours post-infection, there was an additional 0.7-day delay in onset of CPE.ConclusionsA triple combination of tizoxanide, ribavirin, and hydroxychloroquine produced a reduction in SARS-COV-2 viral replication in Vero E6 cells, warranting exploration in additional cell lines as well as human clinical trials.

scholarly journals P38 MAPK Promotes Migration and Metastatic Activity of BRAF Mutant Melanoma Cells by Inducing Degradation of PMCA4b

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1209
Author(s):  
Randa Naffa ◽  
Lisa Vogel ◽  
Luca Hegedűs ◽  
Katalin Pászty ◽  
Sarolta Tóth ◽  
...  
Keyword(s):  
P38 Mapk ◽  
Mapk Pathway ◽  
Melanoma Cells ◽  
Cell System ◽  
Metastasis Suppressor ◽  
Melanoma Metastasis ◽  
P38 Mapk Pathway ◽  
Metastatic Activity ◽  
Braf Mutant

Metastatic melanoma is the most aggressive type of skin cancer. Previously, we identified the plasma membrane Ca2+ pump isoform 4b (PMCA4b or ATP2B4) as a putative metastasis suppressor in BRAF mutant melanoma cells. Metastasis suppressors are often downregulated in cancer, therefore, it is important to identify the pathways involved in their degradation. Here, we studied the role of p38 MAPK in PMCA4b degradation and its effect on melanoma metastasis. We found that activation of p38 MAPK induces internalization and subsequent degradation of PMCA4b through the endo/lysosomal system that contributes to the low PMCA4b steady-state protein level of BRAF mutant melanoma cells. Moreover, BRAF wild type cell models including a doxycycline-inducible HEK cell system revealed that p38 MAPK is a universal modulator of PMCA4b endocytosis. Inhibition of the p38 MAPK pathway markedly reduced migration, colony formation and metastatic activity of BRAF mutant cells in vitro partially through an increase in PMCA4b and a decrease in β4 integrin abundance. In conclusion, our data suggest that the p38 MAPK pathway plays a key role in PMCA4b degradation and inhibition of this pathway—by increasing the stability of PMCA4b—may provide a potential therapeutic target for inhibition of melanoma progression and metastasis.

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