scholarly journals CBMT-07. BIOMARKERS OF AGGRESSIVENESS IN IDH MUTANT GLIOMA

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi34-vi34
Author(s):  
Tomohiro Yamasaki ◽  
Adrian Lita ◽  
Tyrone Dowdy ◽  
Mark Gilbert ◽  
Mioara Larion
Keyword(s):  
Cell Lines ◽  
Imaging Spectroscopy ◽  
Mutant Gene ◽  
Raman Imaging ◽  
Malignant Progression ◽  
Mass Spectrometry Analysis ◽  
Live Cells ◽  
Lower Grade ◽  
Spectrometry Analysis ◽  
Idh Mutations

Abstract BACKGROUND Gliomas with isocitrate dehydrogenase (IDH) mutations in adults evolve from lower-grade gliomas to secondary glioblastomas (GBM), a fatal disease with fast progression. IDH mutation occurs early in tumorigenesis, and persistently contribute to the reprograming of glucose, lipid and amino acid metabolism. This offer a plethora of potential biomarkers of progression. However, because it is extremely difficult to detect the distribution and transfer of metabolites changing in every moment in a single cell, the involvement of metabolites produced by mutant IDH in malignant progression remains understudied. MATERIALS AND METHODS Raman imaging spectroscopy, which can image chemical bonds and concentration of molecules at submicron spatial resolution, enables detection of spatiotemporal changes of metabolomes in live cells. We developed the software called Biomolecular Component Analysis (BCAbox) to deconvolute the recorded raw Raman spectra, leading to detection of unique spectral features of different classes of biomolecules. RESULTS AND CONCLUSIONS We applied Raman imaging spectroscopy to GBM cell lines that were transfected with IDH1 mutant gene. Our results indicated that lipid metabolism has a unique profile in IDH1 mutant gliomas. Subsequent mass spectrometry analysis of extracted organelle revealed the exact classes of lipids altered in the IDH mutant glioma and suggested biomarkers unique to IDH1 mutant. We will report our validation studies of the biomarkers in patient-derived IDH mutant glioma cell lines and patients derived-orthotopic xenograft mouse models with different degrees of aggressiveness and in matched primary versus recurrent gliomas. The results of the present study may provide novel insights into the discovery of metabolic biomarkers for the malignant progression in IDH mutant gliomas.

scholarly journals Cytotoxic Fractions from Hechtia glomerata Extracts and p-Coumaric Acid as MAPK Inhibitors

Molecules ◽  
2021 ◽  
Vol 26 (4) ◽  
pp. 1096
Author(s):  
Tommaso Stefani ◽  
Antonio Romo-Mancillas ◽  
Juan J. J. Carrizales-Castillo ◽  
Eder Arredondo-Espinoza ◽  
Karla Ramírez-Estrada ◽  
...  
Keyword(s):  
Cell Lines ◽  
Active Sites ◽  
High Performance ◽  
Mapk Signaling ◽  
Mass Spectrometry Analysis ◽  
Breast Adenocarcinoma ◽  
Coumaric Acid ◽  
Molecular Docking Study ◽  
Spectrometry Analysis ◽  
Mapk Inhibitors

Preliminary bioassay-guided fractionation was performed to identify cytotoxic compounds from Hechtia glomerata, a plant that is used in Mexican ethnomedicine. Organic and aqueous extracts were prepared from H. glomerata’s leaves and evaluated against two cancer cell lines. The CHCl3/MeOH (1:1) active extract was fractionated, and the resulting fractions were assayed against prostate adenocarcinoma PC3 and breast adenocarcinoma MCF7 cell lines. Active fraction 4 was further analyzed by high-performance liquid chromatography–quadrupole time-of-flight–mass spectrometry analysis to identify its active constituents. Among the compounds that were responsible for the cytotoxic effects of this fraction were flavonoids, phenolic acids, and aromatic compounds, of which p-coumaric acid (p-CA) and its derivatives were abundant. To understand the mechanisms that underlie p-CA cytotoxicity, a microarray assay was performed on PC3 cells that were treated or not with this compound. The results showed that mitogen-activated protein kinases (MAPKs) that regulate many cancer-related pathways were targeted by p-CA, which could be related to the reported effects of reactive oxygen species (ROS). A molecular docking study of p-CA showed that this phenolic acid targeted these protein active sites (MAPK8 and Serine/Threonine protein kinase 3) at the same binding site as their inhibitors. Thus, we hypothesize that p-CA produces ROS, directly affects the MAPK signaling pathway, and consequently causes apoptosis, among other effects. Additionally, p-CA could be used as a platform for the design of new MAPK inhibitors and re-sensitizing agents for resistant cancers.

scholarly journals Optimizing the fragment complementation of APEX2 for detection of specific protein-protein interactions in live cells

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Miaomiao Xue ◽  
Junjie Hou ◽  
Linlin Wang ◽  
Dongwan Cheng ◽  
Jingze Lu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
High Specificity ◽  
Mass Spectrometry Analysis ◽  
Live Cells ◽  
Protein Protein Interactions ◽  
Hek 293 ◽  
Spectrometry Analysis ◽  
Specificity And Sensitivity ◽  
Fragment Complementation

Abstract Dynamic protein-protein interactions (PPIs) play crucial roles in cell physiological processes. The protein-fragment complementation (PFC) assay has been developed as a powerful approach for the detection of PPIs, but its potential for identifying protein interacting regions is not optimized. Recently, an ascorbate peroxidase (APEX2)-based proximity-tagging method combined with mass spectrometry was developed to identify potential protein interactions in live cells. In this study, we tested whether APEX2 could be employed for PFC. By screening split APEX2 pairs attached to FK506-binding protein 12 (FKBP) and the FKBP12-rapamycin binding (FRB) domain, which interact with each other only in the presence of rapamycin, we successfully obtained an optimized pair for visualizing the interaction between FRB and FKBP12 with high specificity and sensitivity in live cells. The robustness of this APEX2 pair was confirmed by its application toward detecting the STIM1 and Orial1 homodimers in HEK-293 cells. With a subsequent mass spectrometry analysis, we obtained five different biotinylated sites that were localized to the known interaction region on STIM1 and were only detected when the homodimer formed. These results suggest that our PFC pair of APEX2 provides a potential tool for detecting PPIs and identifying binding regions with high specificity in live cells.

scholarly journals Suaeda vermiculata Aqueous-Ethanolic Extract-Based Mitigation of CCl4-Induced Hepatotoxicity in Rats, and HepG-2 and HepG-2/ADR Cell-Lines-Based Cytotoxicity Evaluations

Plants ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 1291
Author(s):  
Salman A. A. Mohammed ◽  
Riaz A. Khan ◽  
Mahmoud Z. El-Readi ◽  
Abdul-Hamid Emwas ◽  
Salim Sioud ◽  
...  
Keyword(s):  
Cell Lines ◽  
Radical Scavenging ◽  
Ethanolic Extract ◽  
Mass Spectrometry Analysis ◽  
Male Rat ◽  
Cytotoxicity Test ◽  
Animal Group ◽  
Spectrometry Analysis ◽  
Ic50 Values ◽  
Aqueous Ethanolic Extract

Suaeda vermiculata, an edible halophytic plant, used by desert nomads to treat jaundice, was investigated for its hepatoprotective bioactivity and safety profile on its mother liquor aqueous-ethanolic extract. Upon LC-MS (Liquid Chromatography-Mass Spectrometry) analysis, the presence of several constituents including three major flavonoids, namely quercetin, quercetin-3-O-rutinoside, and kaempferol-O-(acetyl)-hexoside-pentoside were confirmed. The aqueous-ethanolic extract, rich in antioxidants, quenched the DPPH (1,1-diphenyl-2-picrylhydrazyl) radicals, and also showed noticeable levels of radical scavenging capacity in ABTS (2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid) assay. For the hepatoprotective activity confirmation, the male rat groups were fed daily, for 7 days (n = 8/group, p.o.), either carboxyl methylcellulose (CMC) 0.5%, silymarin 200 mg/kg, the aqueous-ethanolic extract of the plant Suaeda vermiculata (100, 250, and 500 mg/kg extract), or quercetin (100 mg/kg) alone, and on day 7 of the administrations, all the animal groups, excluding a naïve (250 mg/kg aqueous-ethanolic extract-fed), and an intact animal group were induced hepatotoxicity by intraperitoneally administering carbon tetrachloride (CCl4). All the animals were sacrificed after 24 h, and aspartate transaminase and alanine transaminase serum levels were observed, which were noted to be significantly decreased for the aqueous-ethanolic extract, silymarin, and quercetin-fed groups in comparison to the CMC-fed group (p < 0.0001). No noticeable adverse effects were observed on the liver, kidney, or heart’s functions of the naïve (250 mg/kg) group. The aqueous-ethanolic extract was found to be safe in the acute toxicity (5 g/kg) test and showed hepatoprotection and safety at higher doses. Further upon, the cytotoxicity testings in HepG-2 and HepG-2/ADR (Adriamycin resistant) cell-lines were also investigated, and the IC50 values were recorded at 56.19 ± 2.55 µg/mL, and 78.40 ± 0.32 µg/mL (p < 0.001, Relative Resistance RR 1.39), respectively, while the doxorubicin (Adriamycin) IC50 values were found to be 1.3 ± 0.064, and 4.77 ± 1.05 µg/mL (p < 0.001, RR 3.67), respectively. The HepG-2/ADR cell-lines when tested in a combination of the aqueous-ethanolic extract with doxorubicin, a significant reversal in the doxorubicin’s IC50 value by 2.77 folds (p < 0.001, CI = 0.56) was noted as compared to the cytotoxicity test where the extract was absent. The mode of action for the reversal was determined to be synergistic in nature indicating the role of the aqueous-ethanolic extract.

scholarly journals MBRS-51. MUTATIONS IN BRPF1 FOUND IN SHH MEDULLOBLASTOMA PREVENT INTERACTION WITH TP53 AND LEADS TO RADIORESISTANCE IN VITRO

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii406-iii407
Author(s):  
Mikaela Vouri ◽  
Audrey Mercier ◽  
Patricia Benites Goncalves da Silva ◽  
Konstantin Okonechnikov ◽  
Antoine Forget ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Apoptotic Cell ◽  
Mutant Gene ◽  
Conditional Knockout ◽  
Mass Spectrometry Analysis ◽  
Cerebellar Granule Neuron ◽  
Spectrometry Analysis ◽  
Shh Pathway

Abstract Medulloblastoma (MB) is one of the most common pediatric tumors in children. Among them, SHH subgroups of MB (MBSHH) is characterized by constitutive activation of SHH pathway. Somatic mutations in BRPF1, a chromatin modifier, is found in more than 5% of MBSHH and accounts for almost 20% of adult MBSHH but its potential role in MBSHH pathophysiology is still unknown. In this study, we first examined the function of Brpf1 on pro-tumorigenic features of MBSHH and evaluated molecular pathways regulated by Brpf1 using Brpf1floxed::Atoh1-Cre conditional knockout mice, in which Brpf1 is conditionally deleted in cerebellar granule neuron progenitors (GNPs). While RNA-seq analysis on GNPs from Brpf1 WT and KO mice showed significant differences in the pathways related with cell cycle and cell death, deletion of Brpf1 did not cause acceleration of tumorigenesis in the Ptch1 heterozygous tumor-prone. Background: Co-immunoprecipitation followed by mass spectrometry analysis identified interaction partners of BRPF1 including MOZ, MORF and ING5, known partners of BRPF1. Gene ontology analysis also depicted pathways important for cell cycle progression, cell death and response to DNA damage. Consistent with these observations, TP53 was identified as a novel co-factor of BRPF1. Of note, some of MBSHH-relevant BRPF1 mutations prevented interaction with TP53. According to the previous finding that cytosolic TP53 is required for apoptotic cell death, GNPs expressing the BRPF1-R600X mutant gene exhibited the resistance to irradiation-induced cell death. In conclusion, our data revealed that BRPF1 mutants found in MBSHH could prevent the complex formation with TP53, leading to enhanced resistance to cell apoptosis.

scholarly journals Development and Initial Characterization of Cellular Models for COG Complex-Related CDG-II Diseases

2021 ◽  
Vol 12 ◽  
Author(s):  
Farhana Taher Sumya ◽  
Irina D. Pokrovskaya ◽  
Vladimir Lupashin
Keyword(s):  
Cell Lines ◽  
Mutant Cell ◽  
Hek293t Cell ◽  
Treatment Guideline ◽  
Mass Spectrometry Analysis ◽  
Wild Type ◽  
Spectrometry Analysis ◽  
Cellular Models ◽  
Cog Complex ◽  
Organ Systems

Conserved Oligomeric Golgi (COG) is an octameric protein complex that orchestrates intra-Golgi trafficking of glycosylation enzymes. Over a hundred individuals with 31 different COG mutations have been identified until now. The cellular phenotypes and clinical presentations of COG-CDGs are heterogeneous, and patients primarily represent neurological, skeletal, and hepatic abnormalities. The establishment of a cellular COG disease model will benefit the molecular study of the disease, explaining the detailed sequence of the interplay between the COG complex and the trafficking machinery. Moreover, patient fibroblasts are not a good representative of all the organ systems and cell types that are affected by COG mutations. We developed and characterized cellular models for human COG4 mutations, specifically in RPE1 and HEK293T cell lines. Using a combination of CRISPR/Cas9 and lentiviral transduction technologies, both myc-tagged wild-type and mutant (G516R and R729W) COG4 proteins were expressed under the endogenous COG4 promoter. Constructed isogenic cell lines were comprehensively characterized using biochemical, microscopy (superresolution and electron), and proteomics approaches. The analysis revealed similar stability and localization of COG complex subunits, wild-type cell growth, and normal Golgi morphology in all three cell lines. Importantly, COG4-G516R cells demonstrated increased HPA-647 binding to the plasma membrane glycoconjugates, while COG4-R729W cells revealed high GNL-647 binding, indicating specific defects in O- and N-glycosylation. Both mutant cell lines express an elevated level of heparin sulfate proteoglycans. Moreover, a quantitative mass-spectrometry analysis of proteins secreted by COG-deficient cell lines revealed abnormal secretion of SIL1 and ERGIC-53 proteins by COG4-G516R cells. Interestingly, the clinical phenotype of patients with congenital mutations in the SIL1 gene (Marinesco-Sjogren syndrome) overlaps with the phenotype of COG4-G516R patients (Saul-Wilson syndrome). Our work is the first compressive study involving the creation of different COG mutations in different cell lines other than the patient’s fibroblast. It may help to address the underlying cause of the phenotypic defects leading to the discovery of a proper treatment guideline for COG-CDGs.

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