P13.09 Inconsistent effect of temozolomide exposure on cell viability in glioblastoma cell line models - a systematic review

2021 ◽  
Vol 23 (Supplement_2) ◽  
pp. ii34-ii34
Author(s):  
M M C Bruce ◽  
M T C Poon ◽  
P M Brennan
Keyword(s):  
Systematic Review ◽  
Cell Viability ◽  
Cell Line ◽  
Cell Lines ◽  
Exposure Duration ◽  
Laboratory Research ◽  
Glioblastoma Cell Line ◽  
Glioblastoma Cell ◽  
Glioblastoma Cell Lines ◽  
Common Cell

Abstract BACKGROUND The alkylating agent temozolomide is part of standard care for patients with glioblastoma. Potential novel therapeutic agents are often first evaluated against temozolomide in glioblastoma cell line models. Despite the importance of this step in compound development, there is no standard concentration or exposure duration of temozolomide in laboratory research, and consistency in the effect of temozolomide on glioblastoma cell lines has not been assessed. This systematic review aimed to summarise the concentration and exposure duration of temozolomide and its effect on cell viability in studies using glioblastoma cell lines. MATERIAL AND METHODS We searched Medline and Embase Jan 1994 - Feb 2021 for studies that used at least one glioblastoma cell line and reported a measure of cell viability associated with temozolomide exposure. Studies were excluded if they used modified cell lines or did not report a cell viability measure associated with temozolomide as monotherapy. One reviewer screened all records and two reviewers assessed potentially eligible studies for inclusion. The main data items included the cell lines used, the concentration and exposure duration to temozolomide, and cell viability measures. We summarised findings using descriptive statistics. RESULTS Of 1,533 potentially eligible studies we included 213 studies reporting 209 different cell lines. The most common cell lines were U87, U251 and T98G, used in 61%, 41%, and 27% of studies, respectively. Twenty-five (12%) studies used patient-derived cell lines. The concentration of temozolomide used ranged from 0 to 8000μM. The temozolomide exposure duration ranged from <24 hours to >96 hours, with 29% studies using 72 hours. The most common cell viability measure was half maximal inhibitory concentration (IC50), which was reported in 183 (86%) studies. The median IC50 in 32 studies using the U87 cell line was 180μM (interquartile range [IQR]: 52–254μM) at 48-hour temozolomide exposure and 202μM (IQR 52–518μM) at 72-hour exposure. The median IC50 in 31 studies using U251 cell line was 84μM (IQR: 34–324μM) at 48-hour exposure and 102μM (IQR: 35–358μM) at 72-hour exposure. CONCLUSION Experimental setup of temozolomide and its effect on cell viability vary widely between studies using similar glioblastoma cell lines. This inconsistency of response to temozolomide questions reproducibility and the translational value of study findings.

DDRE-16. DRUG REPURPOSING SCREEN REVEALS GLIOBLASTOMA CELL LINE SUSCEPTIBILITY TO STATINS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi77-vi78
Author(s):  
Dylan Harwood ◽  
Signe Michaelsen ◽  
Filip Mundt ◽  
Bjarne Kristensen
Keyword(s):  
Cell Viability ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Gene Knockout ◽  
Tumor Resection ◽  
Glioblastoma Cell ◽  
Potential Drug ◽  
Drug Candidates ◽  
Glioblastoma Cell Lines

Abstract BACKGROUND The standard therapy for glioblastoma patients is tumor resection followed by radiotherapy and temozolomide chemotherapy. Although glioblastoma has been extensively molecularly profiled along with other cancers, this knowledge has not yet been translated into improved survival outcomes. We used a bioinformatics approach to identify potential novel therapeutic strategies for glioblastoma. OBJECTIVES: Comprehensive online datasets which have assessed up to 1376 cancer cell lines in multiple ways were interrogated to identify potential drug candidates for glioblastoma. METHODS Datasets included were from the cancer cell line encyclopedia (mRNA expression), the Achilles project (cell viability following Crispr-Cas9 knockout) and PRISM (drug treatment). A t-test comparing cell viability of glioblastoma cell lines versus other cancers was used to identify potential drug candidates, followed by the use of multiple statistical tools to investigate potential mechanism of action and status of biomarkers. RESULTS Fluvastatin and pitavastatin produced the most significant effects in glioblastoma cell lines. The anti-cancer properties of statins have previously been attributed to the inhibition of HMG-Coa reductase. Here, we found their effects correlated with erastin, an enhancer of ferroptosis and with gene knockout of UBIAD1, which participates in non-mitochondrial ubiquinone synthesis. These effects were both found in glioblastoma cells and other cancers with a mesenchymal-like phenotype. CONCLUSION Statins appeared to be especially effective against glioblastoma lines and the effect could be linked to ferroptosis and inhibition of UBIAD1. In vitro validation of this finding is ongoing.

Impact of Zika virus infection in human glioblastoma cell lines.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e13563-e13563
Author(s):  
Andre P. Fay
Keyword(s):  
Cell Migration ◽  
Cell Adhesion ◽  
Cell Viability ◽  
Cell Lines ◽  
Zika Virus ◽  
Cell Infiltration ◽  
Glioblastoma Cell ◽  
Human Glioblastoma ◽  
Human Glioblastoma Cell ◽  
Glioblastoma Cell Lines

e13563 Background: Glioblastoma (GBM) is the most common central nervous system (CNS) tumor. Despite available therapies survival remains poor, and new treatment strategies are needed. Oncolytic viral therapy is under investigation in GBM. Zika virus (ZV) infection has demonstrated inhibition of neuronal precursor cells proliferation in preclinical studies. This study aims to evaluate the effects of ZV infection on human glioblastoma cell lines survival. Methods: Two GBM cell lines (U138 and U 251) were infected by 2 hours with ZV using the titer of PFU/ml diluted in 1:100, 1:1000, 1: 10000. Cell. We performed tests to evaluate cell viability by MTT and protease active assay, cell migration trough a wound healing assay and cell infiltration using insert culture method. Also the cell lines were tested by cell adhesion capacity, apoptosis (Caspase 3/7), interleukins levels and cell surface markers for CD 14 and CD73.These results were compared to controls. Results: Our study has demonstrated a reduction in cell viability in U138 lineage trough MTT assay. In the U251 lineage and in the others tests for cytotoxicity/viability, ZK did not altered cell viability neither cell migration compared to controls. We showed that ZV caused reduction in cell invasion and resulted in increased rates of apoptosis in both cell lines. We observed that infection of ZV caused increased rates of cell adhesion and CD73 marker. Conclusions: These findings suggest that ZV infection may be associated with increased CD 73 Expression thus increasing cell adhesion and cell infiltration. The ZV may be cause an increase rates of apoptosis and influencing cell cytotoxicity and viability. Further investigations to explore the role of ZV in GBM treatment are warranted.

scholarly journals RDNA-14. RADIATION-INDUCED miR-4516 CONTRIBUTES TO RADIO-RESISTANCE AND PROMOTES AGGRESSIVE PHENOTYPE IN GLIOBLASTOMA

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi209-vi210
Author(s):  
Ebin Sebastian ◽  
Tiantian Cui ◽  
Erica Hlavin Bell ◽  
Joseph McElroy ◽  
Benjamin Johnson ◽  
...  
Keyword(s):  
Brain Tumor ◽  
Cell Viability ◽  
Cell Lines ◽  
Poor Prognosis ◽  
Radiation Treatment ◽  
Critical Role ◽  
Luciferase Reporter ◽  
Glioblastoma Cell ◽  
Clonogenic Assays ◽  
Glioblastoma Cell Lines

Abstract BACKGROUND Glioblastoma is the most aggressive brain tumor with poor prognosis despite the best available treatment. MicroRNAs (miRNAs) are emerging as promising, novel prognostic biomarkers and therapeutic targets in glioblastoma. In a previous study, we demonstrated that miR-4516 predicts poor prognosis and functions as an oncogene in glioblastoma. Aim of the current study is to examine the role miR-4516 in radiation resistance and identify downstream targets contributing to this phenotype METHODS Radiosensitization was evaluated by cell viability and clonogenic assays. Cell apoptosis was evaluated using flow cytometry and immunoblotting. Potential targets of miR-4516 were identified using bioinformatic analysis (Targetscan and miRDB) and confirmed by luciferase reporter assays. Results were validated using immunoblotting. miR-4516 expression in glioblastoma cell lines after radiation treatment was quantified by qRT-PCR. RESULTS Expression of miR-4516 was increased up to 15 fold following radiation treatment, peaking at around 15min-60 min in primary and established glioblastoma cell lines including GBM 08-387, GBM 30 and U87-MG. Furthermore, inhibition of miR-4516 sensitized GBM 08-387, GBM30 and U87-MG cells to radiation in comparison to control groups as determined by cell viability and clonogenic assays. Further, miR-4516 inhibition induced apoptosis in these cell lines following radiation treatment. While conducting mechanistic studies, we found that the tumor-promoting function of miR-4516 was, in part, mediated by inhibition of p21 and PTPN14, two direct targets of miR-4516 CONCLUSION Our data suggest that radiation induces the expression of miR-4516 in glioblastoma cell lines. This miRNA plays a critical role in radio-resistance and promotes aggressive phenotypes in glioblastoma and therefore, functional analyses of its target pathways may uncover novel therapeutically vulnerable target(s) in glioblastoma. FUNDING: R01CA108633, R01CA169368, RC2CA148190, U10CA180850-01(NCI), Brain Tumor Funders Collaborative Grant, and OSU-CCC (all to AC). The Ton and Patricia Bohnenn Fund for Neuro_Oncology Research (to PR).

scholarly journals Long Non-Coding RNA MALAT1 Decreases the Sensitivity of Resistant Glioblastoma Cell Lines to Temozolomide

2017 ◽  
Vol 42 (3) ◽  
pp. 1192-1201 ◽  
Author(s):  
Hongwei Li ◽  
Xiaoli Yuan ◽  
Dongming Yan ◽  
Dongpeng Li ◽  
Fangxia Guan ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Viability ◽  
Cell Lines ◽  
Epithelial Mesenchymal Transition ◽  
Tumor Biology ◽  
Glioblastoma Cell ◽  
Mesenchymal Transition ◽  
Non Coding Rna ◽  
Long Non Coding Rna ◽  
Glioblastoma Cell Lines

Background/Aim: Multidrug resistance (MDR) is largely responsible for the failure of chemotherapy. The long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript (MALAT1) has been reported to be closely related to tumor biology. In the present study, whether MALAT1 contributes to the resistance of glioblastoma cell lines to temozolomide (TMZ) was investigated. Methods: The glioblastoma cell lines U251 and U87 were exposed to increasing concentrations of TMZ to generate TMZ-resistant colonies (the U251/TMZ and U87/TMZ cell lines). The expression levels of MALAT1 and proteins related to epithelial-mesenchymal transition (EMT) were detected by real-time PCR and western blot, respectively. After the transfection of si-MALAT1 or pcDNA-MALAT1, cell viability, mRNA expression of MDR-associated proteins (MDR1, MRP5 and LRP1), and protein expression of EMT related proteins (ZEB1, Snail and SLUG) were evaluated. Results: The expression of MALAT1 was upregulated in the U251/TMZ and U87/TMZ cell lines compared to that in U251 and U87 cell lines, respectively. The treatment of si-MALAT1 decreased MDR1, MRP5, and LRP1 expression, enhanced cell sensitivity to TMZ, and downregulated ZEB1 protein expression, whereas pcDNA-MALAT1 had the opposite effects. However, the effects of si-MALAT1 on MDR -associated protein expression, cell viability, and EMT status were reversed by the transfection of pcDNA-ZEB1, and the effects of pcDNA-MALAT1 were reversed by the transfection of si-ZEB1. In vivo, MALAT1 overexpression enhanced tumors’ TMZ resistance and upregulated ZEB1 expression. Conclusion: MALAT1 decreased the sensitivity of resistant glioma cell lines to TMZ by regulating ZEB1.

scholarly journals Circumdatin-Aspyrone Conjugates from the Coral-Associated Aspergillus ochraceus LCJ11-102

Marine Drugs ◽  
2019 ◽  
Vol 17 (7) ◽  
pp. 400 ◽  
Author(s):  
Yaqin Fan ◽  
Yalin Zhou ◽  
Yuqi Du ◽  
Yi Wang ◽  
Peng Fu ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Nucleophilic Addition ◽  
Human Cancer ◽  
Aspergillus Ochraceus ◽  
Glioblastoma Cell Line ◽  
Glioblastoma Cell ◽  
Human Cancer Cell ◽  
Human Cancer Cell Lines

Ochrazepines A−D (1−4), four new conjugates dimerized from 2-hydroxycircumdatin C (5) and aspyrone (6) by a nucleophilic addition to epoxide, were isolated from the fermentation broth of the coral-associated Aspergillus ochraceus strain LCJ11-102. Their structures including absolute configurations were determined based on spectroscopic analysis and chemical methods. Compounds 1−4 were also obtained by the semisynthesis from a nucleophilic addition of 2-hydroxycircumdatin C (5) to aspyrone (6). New compound 1 exhibited cytotoxic activity against 10 human cancer cell lines while new compounds 2 and 4 selectively inhibited U251 (human glioblastoma cell line) and compound 3 was active against A673 (human rhabdomyoma cell line), U87 (human glioblastoma cell line), and Hep3B (human liver cancer cell line) with IC50 (half maximal inhibitory concentration) values of 2.5–11.3 μM among 26 tested human cancer cell lines.

scholarly journals The lipid phosphatase activity of PTEN is critical for stabilizing intercellular junctions and reverting invasiveness

2001 ◽  
Vol 155 (7) ◽  
pp. 1129-1136 ◽  
Author(s):  
Larissa Kotelevets ◽  
Jolanda van Hengel ◽  
Erik Bruyneel ◽  
Marc Mareel ◽  
Frans van Roy ◽  
...  
Keyword(s):  
Cell Line ◽  
Phosphatase Activity ◽  
Cell Lines ◽  
Dependent Manner ◽  
Glioblastoma Cell ◽  
Wild Type ◽  
Invasive Phenotype ◽  
Lipid Phosphatase ◽  
Glioblastoma Cell Lines ◽  
E Cadherin

To analyze the implication of PTEN in the control of tumor cell invasiveness, the canine kidney epithelial cell lines MDCKras-f and MDCKts-src, expressing activated Ras and a temperature-sensitive v-Src tyrosine kinase, respectively, were transfected with PTEN expression vectors. Likewise, the human PTEN-defective glioblastoma cell lines U87MG and U373MG, the melanoma cell line FM-45, and the prostate carcinoma cell line PC-3 were transfected. We demonstrate that ectopic expression of wild-type PTEN in MDCKts-src cells, but not expression of PTEN mutants deficient in either the lipid or both the lipid and protein phosphatase activities, reverted the morphological transformation, induced cell–cell aggregation, and suppressed the invasive phenotype in an E-cadherin–dependent manner. In contrast, overexpression of wild-type PTEN did not counteract Ras-induced invasiveness of MDCKras-f cells expressing low levels of E-cadherin. PTEN effects were not associated with marked changes in accumulation or phosphorylation levels of E-cadherin and associated catenins. Wild-type, but not mutant, PTEN also reverted the invasive phenotype of U87MG, U373MG, PC-3, and FM-45 cells. Interestingly, PTEN effects were mimicked by N-cadherin–neutralizing antibody in the glioblastoma cell lines. Our data confirm the differential activities of E- and N-cadherin on invasiveness and suggest that the lipid phosphatase activity of PTEN exerts a critical role in stabilizing junctional complexes and restraining invasiveness.

Heterogeneity and modulation of tumor-associated antigens in human glioblastoma cell lines

1989 ◽  
Vol 71 (3) ◽  
pp. 388-397 ◽  
Author(s):  
Marco Colombatti ◽  
Bruno Dipasquale ◽  
Lorena Del-l'Arciprete ◽  
Massimo Gerosa ◽  
Giuseppe Tridente
Keyword(s):  
Cell Surface ◽  
Cell Line ◽  
Cell Lines ◽  
Glioblastoma Cell ◽  
Heterogeneous Distribution ◽  
Human Glioblastoma ◽  
Human Glioblastoma Cell ◽  
Tumor Associated Antigens ◽  
Glioblastoma Cell Lines

✓ Seven human glioblastoma cell lines established in vitro from primary tumor explants were studied. A marked heterogeneity of glial fibrillary acidic protein was observed whereas vimentin was uniformly expressed by all cell lines. Indirect immunofluorescence and flow cytofluorometry revealed a heterogeneous distribution of surface GE 2 and CG 12 tumor-associated antigens (TAA's): three cell lines were positive (> 69% TAA-positive cells) and three cell lines were negative (< 9% TAA-positive cells). One cell line (Hu 228) was moderately positive at early culture passages and subsequently acquired a TAA-negative phenotype. The difference in the relative amounts of surface TAA's of the three positive cell lines was less than twofold. In spite of the heterogeneous distribution of surface TAA's, all cell lines exhibited considerable amounts of intracellular TAA. Treatment with phorbol esters and density-dependent growth arrest decreased the percentage of the TAA-positive cells and the amount of cell-surface TAA's in one cell line (Hu 195). Interferon-γ treatment in vitro increased the percentage of CG 12-positive cells by 12% and the amount of cell-surface CG 12 antigens by 38% as compared to untreated cells. The percentage of TAA-positive cells among phorbol ester-treated cells of the Hu 195 cell line was lowest 48 hours after treatment, but returned to normal values within the next 48 hours. Reduction of 3H-thymidine incorporation preceded the decrease in number of TAA-positive cells by about 18 hours. Two-color fluorescence analysis performed in positive cell lines for simultaneous determination of surface TAA's and deoxyribonucleic acid content or reactivity with the proliferation-associated Ki67 intracellular marker indicated that GE 2 and CG 12 antigens are expressed preferentially by actively proliferating glioma cells. The results of this study indicate the existence of two different phenotypes in cultured human glioblastoma cells: surface TAA-positive/cytosol TAA-positive and surface TAA-negative/cytosol TAA-positive cell populations. In addition, modulation of TAA expression was dependent on the cell-cycle differentiation stage, culture conditions, and proliferative state of the cells.

scholarly journals Multiple Localization of Endogenous MARK4L Protein in Human Glioma

2009 ◽  
Vol 31 (5) ◽  
pp. 357-370
Author(s):  
Ivana Magnani ◽  
Chiara Novielli ◽  
Melissa Bellini ◽  
Gaia Roversi ◽  
Lorenzo Bello ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Glioma Cell ◽  
Cellular Localization ◽  
Protein Identification ◽  
Glioblastoma Cell Line ◽  
Glioblastoma Cell ◽  
Human Glioma ◽  
Glioma Cell Lines ◽  
Biochemical Fractionation

Background: We have previously shown that the sustained expression of MARK4L transcripts in glioma and neural progenitors (NHNPs) declines after exposure to antisense MARK4L oligonucleotides in glioblastoma cell lines. Array-CGH confirmed the genomic duplication of MARK4L identified by FISH in a glioblastoma cell line. This background together with literature data on the exogenous association of MARK4 with interphase centrosome prompted us to investigate the sub-cellular localization of the endogenous MARK4L protein aiming at achieving insights on its possible role in the pathomechanisms of glioma.Methods: Immunodetection was carried out to validate the specificity of MARK4L antibody in gliomas and NHNPs. Mass spectrometry was applied for MARK4L protein identification in a representative glioblastoma cell line. Combined biochemical fractionation and immunodetection analyses were performed to confirm the sub-cellular localization of MARK4L achieved by immunofluorescence in glioma cell lines.Results: By assigning MARK4L protein within the band immunoprecipitated by the specific antibody we validated our anti-MARK4L antibody. We demonstrated that the endogenous MARK4L: (i) colocalizes with centrosomes at all mitotic stages and resides in centrosome-enriched fractions; (ii) associates with the nucleolus and the midbody and respective fractions, and (iii) co-stains the aberrant centrosome configurations observed in glioma cell lines.Conclusions: The overall data merge on the multiplex entry of MARK4L into the cell cycle and link it to the aberrant centrosomes in glioma cell lines suggesting a possible role of this kinase in the abnormal mitotic processes of human glioma.

scholarly journals Assessment of Intratumoral Heterogeneity in Isolated Human Primary High-Grade Glioma: Cluster of Differentiation 133 and Cluster of Differentiation 15 Double Staining of Glioblastoma Subpopulations

2021 ◽  
Vol 9 (A) ◽  
pp. 87-94
Author(s):  
Ahmad Faried ◽  
Wahyu Widowati ◽  
Rizal Rizal ◽  
Hendrikus M. B. Bolly ◽  
Danny Halim ◽  
...  
Keyword(s):  
Brain Tumors ◽  
Cell Line ◽  
Cell Lines ◽  
Glioblastoma Cell Line ◽  
Glioblastoma Cell ◽  
Double Staining ◽  
Glioblastoma Cells ◽  
Ki 67 ◽  
Primary Glioblastoma ◽  
Cluster Of Differentiation

BACKGROUND: Gliomas are the most common primary brain tumors, representing 50–60% of malignant primary brain tumors. Gliomas are highly heterogeneous with marked inter- and intratumoral diversity. Gliomas heterogeneity is a challenging issue in the development of personalized treatment. The simplest method for studying heterogeneity is using ex vivo cell cultures; in our case, the cell lines were isolated from patient with glioblastomas. AIM: Here, we reported distinct cell subpopulations heterogeneity in glioblastoma cells. METHODS: Human glioblastoma cells isolation is conducted by enzymatic method with combination of collagenase I, hyaluronidase, and trypsin enzyme in proportional amount from patient. Immunostaining was performed to assess glial fibrillary acidic protein (GFAP), Ki-67, isocitrate dehydrogenase-1 (IDH-1) status, and program death ligand-1 (PD-L1) expression. Primary glioblastoma cell line was characterized by flow cytometry (fluorescence-activated cell sorting) analysis based on cluster of differentiation (CD) 133 and CD15 marker expression. U87MG and CGNH-89 cell lines were used as control. Distinct subpopulation analysis was performed by double staining of CD133 and CD15 in isolated primary glioblastoma cell line and its comparative control cells. RESULTS: Our isolated glioblastoma cells morphology was adherent cells which were able to form spheres depending on environment. Immunostaining confirmed GFAP, Ki-67, IDH-1 mutants, and PD-L1 expression. Our isolated glioblastoma cells expressed CD133 and CD15, coexpressed CD133/CD15 in different patterns. The highest subpopulation in primary glioblastoma was CD133+/CD15+. CONCLUSION: Glioblastoma cells can be isolated using enzymatic methods. Isolated glioblastoma cells consist of four different subpopulations distinguished by CD133/CD15 double staining. Intratumoral heterogeneity exists and directly or indirectly depends on their microenvironment.

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