scholarly journals Temporal Quantitative Proteomics Reveals Proteomic and Phosphoproteomic Alterations Associated with Adaptive Response to Hypoxia in Melanoma Cells

Cancers ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 2175
Author(s):  
Keshava K. Datta ◽  
Parthiban Periasamy ◽  
Sonali V. Mohan ◽  
Rebekah Ziegman ◽  
Harsha Gowda
Keyword(s):  
Protein Expression ◽  
Cell Survival ◽  
Cancer Cells ◽  
Cell Lines ◽  
Melanoma Cell ◽  
Adaptive Response ◽  
Quantitative Proteomics ◽  
Cellular Response ◽  
Metabolic Reprogramming ◽  
Melanoma Cell Lines

Hypoxia is a common feature in various solid tumours, including melanoma. Cancer cells in hypoxic environments are resistant to both chemotherapy and radiation. Hypoxia is also associated with immune suppression. Identification of proteins and pathways that regulate cancer cell survival in hypoxic environments can reveal potential vulnerabilities that can be exploited to improve the efficacy of anticancer therapies. We carried out temporal proteomic and phosphoproteomic profiling in melanoma cell lines to identify hypoxia-induced protein expression and phosphorylation changes. By employing a TMT-based quantitative proteomics strategy, we report the identification and quantitation of >7000 proteins and >10,000 phosphosites in melanoma cell lines grown in hypoxia. Proteomics data show metabolic reprogramming as one of the prominent adaptive responses in hypoxia. We identify several novel hypoxia-mediated phosphorylation changes that have not been reported before. They reveal kinase signalling pathways that are potentially involved in modulating cellular response to hypoxia. In addition to known protein expression changes, we identify several novel proteomic alterations associated with adaptive response to hypoxia. We show that cancer cells require the ubiquitin–proteasome system to survive in both normoxia and hypoxia. Inhibition of proteasome activity affects cell survival and may provide a novel therapeutic avenue to target cancer cells in hypoxia. Our study can serve as a valuable resource to pursue novel candidates to target hypoxia in cancers and improve the efficacy of anticancer therapies.

Inhibition of glucose metabolism through treatment of BRAF mutated metastatic melanoma with vemurafenib.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e21005-e21005 ◽  
Author(s):  
Govind Warrier ◽  
Lilibeth Lanceta ◽  
Yoannis Imbert-Fernandez ◽  
Jason Alan Chesney
Keyword(s):  
Glucose Metabolism ◽  
Protein Expression ◽  
Metastatic Melanoma ◽  
Cell Lines ◽  
Melanoma Cell ◽  
Control Point ◽  
Braf Inhibitor ◽  
Growth And Survival ◽  
Metastatic Melanoma Cell ◽  
Melanoma Cell Lines

e21005 Background: Increased glucose metabolism is a hallmark of neoplastic cells that allows self-promotion of growth and survival. The enzyme 6-phosphofructo-2-kinase (PFKFB3) is an integral controller of glycolysis by promoting the synthesis of fructose 2,6-bisphosphonate (F2,6BP) which activates 6-phoshofructo-1-kinase (PFK-1), a rate-limiting enzyme and essential control point in the glycolytic pathway. Additionally, mitogen-activated protein kinase (MAPK) is a key signaling pathway in a number of cancers with mutations of the BRAF component, described most commonly in melanoma, resulting in constitutive activation of the MAPK pathway. We aim to demonstrate that vemurafenib, a BRAF inhibitor, has antiglycolytic activity in sensitive melanoma cell lines which may help guide development of future therapies with specific attention to PFKFB3 as a potential enzymatic target to decrease glycolytic flux thereby inhibiting tumor growth and survival. Methods: Vemurafenib sensitive and resistant variants of two separate human metastatic melanoma cell lines (451Lu and WM983) were treated with 3 mM Vemurafenib for 24 and 48 hours. Additionally, cells from aforementioned lines were probed for PFKFB3 after 24 hours of treatment with vemurafenib. Glycolysis was measured by incubating cells in complete media containing 1 mCi [5-3H]glucose for 60 minutes. [3H]H2O produced by glycolysis through enolase was measured. Results: A decrease in PFKFB3 protein expression was found in vemurafenib sensitive cells compared to controls but not in resistant cells after 24h treatment with 3 mM vemurafenib in both 451Lu and WM983 metastatic melanoma cell lines (n = 2). Treatment with vemurafenib led to decrease in glycolysis compared to untreated controls in both vemurafenib sensitive metastatic melanoma cell lines but not in resistant cell lines (n = 5). Additionally, there was a significant reduction in glycolysis in vemurafenib resistant WM983 at 48 hours compared to resistant untreated control. Conclusions: BRAF mutated metastatic melanoma cells showed decrease in PFKFB3 protein expression and decreased glycolysis after treatment with BRAF inhibitor vemurafenib. Future studies will focus on assessing metastatic melanoma cell viability and glycolytic activity after treatment with combination BRAF inhibition and PFKFB3 specific inhibition.

scholarly journals Targeting Mitochondria in Melanoma

Biomolecules ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 1395
Author(s):  
Sepideh Aminzadeh-Gohari ◽  
Daniela D. Weber ◽  
Luca Catalano ◽  
René G. Feichtinger ◽  
Barbara Kofler ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Cell Lines ◽  
Melanoma Cell ◽  
Mitochondrial Respiration ◽  
Melanoma Cells ◽  
Dermal Fibroblasts ◽  
Glycolytic Activity ◽  
Anti Cancer ◽  
Cancer Agent ◽  
Melanoma Cell Lines

Drastically elevated glycolytic activity is a prominent metabolic feature of cancer cells. Until recently it was thought that tumor cells shift their entire energy production from oxidative phosphorylation (OXPHOS) to glycolysis. However, new evidence indicates that many cancer cells still have functional OXPHOS, despite their increased reliance on glycolysis. Growing pre-clinical and clinical evidence suggests that targeting mitochondrial metabolism has anti-cancer effects. Here, we analyzed mitochondrial respiration and the amount and activity of OXPHOS complexes in four melanoma cell lines and normal human dermal fibroblasts (HDFs) by Seahorse real-time cell metabolic analysis, immunoblotting, and spectrophotometry. We also tested three clinically approved antibiotics, one anti-parasitic drug (pyrvinium pamoate), and a novel anti-cancer agent (ONC212) for effects on mitochondrial respiration and proliferation of melanoma cells and HDFs. We found that three of the four melanoma cell lines have elevated glycolysis as well as OXPHOS, but contain dysfunctional mitochondria. The antibiotics produced different effects on the melanoma cells and HDFs. The anti-parasitic drug strongly inhibited respiration and proliferation of both the melanoma cells and HDFs. ONC212 reduced respiration in melanoma cells and HDFs, and inhibited the proliferation of melanoma cells. Our findings highlight ONC212 as a promising drug for targeting mitochondrial respiration in cancer.

Signatures of protein expression revealed by secretome analyses of cancer associated fibroblasts and melanoma cell lines

2018 ◽  
Vol 174 ◽  
pp. 1-8 ◽  
Author(s):  
Tarcísio Liberato ◽  
Dayelle S. Pessotti ◽  
Isabella Fukushima ◽  
Eduardo S. Kitano ◽  
Solange M.T. Serrano ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Lines ◽  
Melanoma Cell ◽  
Cancer Associated Fibroblasts ◽  
Melanoma Cell Lines

scholarly journals Peroxidasin protein expression and enzymatic activity in metastatic melanoma cell lines are associated with invasive potential

Redox Biology ◽  
2021 ◽  
pp. 102090
Author(s):  
Martina Paumann-Page ◽  
Nikolaus F. Kienzl ◽  
Jyoti Motwani ◽  
Boushra Bathish ◽  
Louise N. Paton ◽  
...  
Keyword(s):  
Protein Expression ◽  
Enzymatic Activity ◽  
Metastatic Melanoma ◽  
Cell Lines ◽  
Melanoma Cell ◽  
Invasive Potential ◽  
Metastatic Melanoma Cell ◽  
Melanoma Cell Lines

scholarly journals CHARACTERISTICS OF MULTIDRUG RESISTANCE IN HUMAN SKIN MELANOMA CELL LINES

2015 ◽  
Vol 14 (4) ◽  
pp. 39-44
Author(s):  
D. A. Afanasieva ◽  
M. A. Baryshnikova ◽  
T. N. Zabotina ◽  
A. A. Borunova ◽  
O. S. Burova ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Cell Line ◽  
Human Skin ◽  
Cell Lines ◽  
Melanoma Cell ◽  
Mdr1 Gene ◽  
Multi Drug Resistance ◽  
Cytotoxic Action ◽  
Skin Melanoma ◽  
Melanoma Cell Lines

MDR is the main obstacle to chemotherapy efficiency. MDR can grow in cancer cells even if only the one cytostatic agent will act. The aim of the nowadays work is to characterize MDR in metastatic human skin melanoma cell lines prepared in “N.N. Blokhin Russian Cancer Research Center”. pgpl70 expression was detected by immunofluorescence methods. mRNA of MDR gene was identified by Reverse Transcriptase- PCR( RT-PCR) method. Rhodamine 123 (Rhl23) emission has been evaluated by flow cyto- fluorimetiy, cytotoxic activity was estimated by MTT-tests. The cells sensitivity to Aianoza cytostatic effects has showed that mel Kor cells were sensitive to Aranoza acting, but mel Ibr and mel Mtp X were not. Mel Ibr cells had expressed pgpl70 from 35 to 50 per cent, it was detected by immunofluorescence reaction. Mel Kor and mel Mtp-X cells were not expressed P-glycoprotein. mRNA of genes responsible for multi-drug resistance - MDR1, BCRP, MRP1 and LRP (MVP) - were detected by PCR. mRNA of BCRP and MRP1 genes has low expression, barely visible stripes after 33 cycles in all cell lines samples. LRP (MVP) genes expression of mRNA, unfortunately, never managed to see. YB1 gene mRNA expression is well, it is typically for cancer cells. mRNA of gene was found in mel MtpX and mel Ibr subclones cell lines. Mel Kor cells didn't contain mRNA of MDR1 gene. The study of the Rhl23 emission from cells showed that mel Kor control cells had accumulated Rhl23 and didn't throw it out. Mel Ibr cell line accumulated Rhl23 and threw out the half part of it. Mel MtpX cell tine had accumulated the less part of Rhl23 and almost all were thrown out. Thus, the study shows that mel Kor cell tine that are sensitive to Aranoza doesn't express pgpl70, not contain mRNA of multi-chug resistance genes and does not throw Rhl23. Mel Ibr cells resistant to the Aranoza cytotoxic action express pgpl70 ,contain mRNA of MDR1 gene and throw out Rhl23. However, mel MtpX cell line resistant to Aranoza does not express pgpl70, but contains mRNA of MDR1 gene and actively throws out Rhl23.

scholarly journals Carbon ion irradiation abrogates Lin28B-induced X-ray resistance in melanoma cells

2017 ◽  
Vol 58 (6) ◽  
pp. 765-771 ◽  
Author(s):  
Seong-Joon Park ◽  
Kyu Heo ◽  
Chulwon Choi ◽  
Kwangmo Yang ◽  
Akiko Adachi ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Cell Lines ◽  
Melanoma Cell ◽  
Ion Irradiation ◽  
Ion Beam ◽  
Melanoma Cells ◽  
Micro Rna ◽  
Carbon Ion ◽  
X Ray ◽  
Melanoma Cell Lines

Abstract The Lin28/let-7 axis plays an important role in tumor initiation and developmental processes. Lin28B is upregulated in a variety of cancers, and its overexpression enhances cancer cell proliferation and radioresistance through the suppression of let-7 micro RNA expression. In this study, we investigated the role of the Lin28/let7 axis as a target for radiosensitization of melanoma cancer cells. The overexpression of Lin28B reduced mature let-7 microRNA expression in melanoma cell lines, and enhanced the sphere-forming ability of melanoma cell lines, which is a characteristic of cancer stem cell (CSC) populations. Interestingly, Lin28B-overexpressed melanoma cells were more resistant to X-ray irradiation than control cells, and Lin28B-induced radioresistance was abolished after carbon ion irradiation. Consistent with these results, Lin28B overexpression reduced the numbers of γH2A.X foci after X-ray irradiation, whereas carbon ion irradiation had no such effect. Our results suggest that a carbon ion beam is more effective than an X-ray beam in terms of killing cancer cells, possibly due to elimination of CSC populations.

scholarly journals Inhibition of the key metabolic pathways, glycolysis and lipogenesis, of oral cancer by bitter melon extract

2019 ◽  
Vol 17 (1) ◽  
Author(s):  
Subhayan Sur ◽  
Hiroshi Nakanishi ◽  
Colin Flaveny ◽  
Joseph E. Ippolito ◽  
Jane McHowat ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Oral Cancer ◽  
Protein Expression ◽  
Cancer Cells ◽  
Cell Lines ◽  
Metabolic Pathways ◽  
Metabolic Reprogramming ◽  
Bitter Melon ◽  
Shotgun Lipidomics ◽  
Oral Cancer Cells

Abstract Background Metabolic reprogramming is one of the hallmarks of cancer which favours rapid energy production, biosynthetic capabilities and therapy resistance. In our previous study, we showed bitter melon extract (BME) prevents carcinogen induced mouse oral cancer. RNA sequence analysis from mouse tongue revealed a significant modulation in “Metabolic Process” by altering glycolysis and lipid metabolic pathways in BME fed group as compared to cancer group. In present study, we evaluated the effect of BME on glycolysis and lipid metabolism pathways in human oral cancer cells. Methods Cal27 and JHU022 cells were treated with BME. RNA and protein expression were analysed for modulation of glycolytic and lipogenesis genes by quantitative real-time PCR, western blot analyses and immunofluorescence. Lactate and pyruvate level was determined by GC/MS. Extracellular acidification and glycolytic rate were measured using the Seahorse XF analyser. Shotgun lipidomics in Cal27 and JHU022 cell lines following BME treatment was performed by ESI/ MS. ROS was measured by FACS. Results Treatment with BME on oral cancer cell lines significantly reduced mRNA and protein expression levels of key glycolytic genes SLC2A1 (GLUT-1), PFKP, LDHA, PKM and PDK3. Pyruvate and lactate levels and glycolysis rate were reduced in oral cancer cells following BME treatment. In lipogenesis pathway, we observed a significant reduction of genes involves in fatty acid biogenesis, ACLY, ACC1 and FASN, at the mRNA and protein levels following BME treatment. Further, BME treatment significantly reduced phosphatidylcholine, phosphatidylethanolamine, and plasmenylethanolamine, and reduced iPLA2 activity. Additionally, BME treatment inhibited lipid raft marker flotillin expression and altered its subcellular localization. ER-stress associated CHOP expression and generation of mitochondrial reactive oxygen species were induced by BME, which facilitated apoptosis. Conclusion Our study revealed that bitter melon extract inhibits glycolysis and lipid metabolism and induces ER and oxidative stress-mediated cell death in oral cancer. Thus, BME-mediated metabolic reprogramming of oral cancer cells will have important preventive and therapeutic implications along with conventional therapies. Graphical abstract

scholarly journals Expression of Potential Targets for Cell-Based Therapies on Melanoma Cells

Life ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 269
Author(s):  
Sophia B. Strobel ◽  
Devayani Machiraju ◽  
Ingrid Hülsmeyer ◽  
Jürgen C. Becker ◽  
Annette Paschen ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Lines ◽  
Melanoma Cell ◽  
Specific Antigen ◽  
Flow Cytometric Analysis ◽  
Membrane Expression ◽  
Primary Tumors ◽  
Cell Therapies ◽  
Melanoma Cell Lines

Tumor antigen-specific redirection of cytotoxic T cells (CTLs) or natural killer (NK) cells including chimeric antigen receptor (CAR-) and T cell receptor (TCR-) cell therapy is currently being evaluated in different tumor entities including melanoma. Expression of melanoma-specific antigen recognized by the respective CAR or TCR directly or presented by HLA molecules is an indispensable prerequisite for this innovative therapy. In this study, we investigated in 168 FFPE tumor specimens of patients with stage I-IV melanoma the protein expression of HER2, TRP2, ABCB5, gp100, p53, and GD2 by immunohistochemistry (IHC). These results were correlated with clinical parameters. Membrane expression of HER2 and GD2 was also investigated in ten melanoma cell lines by flow cytometry for which corresponding tumors were analyzed by IHC. Our results demonstrated that gp100 was the most frequently overexpressed protein (61%), followed by TRP2 (50%), GD2 (38%), p53 (37%), ABCB5 (17%), and HER2 (3%). TRP2 expression was higher in primary tumors compared to metastases (p = 0.005). Accordingly, TRP2 and ABCB5 expression was significantly associated with lower tumor thickness of the primary (p = 0.013 and p = 0.025). There was no association between protein expression levels and survival in advanced melanoma patients. Flow cytometric analysis revealed abundant surface expression of GD2 and HER2 in all melanoma cell lines. The discordant HER2 expression in situ and in vitro suggests a tissue culture associated induction. In summary, our data support the use of gp100 and GD2 as a potential target for developing engineered TCR- or CAR-cell therapies, respectively, against melanoma.

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