scholarly journals Disruption of the Complex between GAPDH and Hsp70 Sensitizes C6 Glioblastoma Cells to Hypoxic Stress

2021 ◽  
Vol 22 (4) ◽  
pp. 1520
Author(s):  
Marina A. Mikeladze ◽  
Elizaveta A. Dutysheva ◽  
Victor G. Kartsev ◽  
Boris A. Margulis ◽  
Irina V. Guzhova ◽  
...  
Keyword(s):  
Cell Death ◽  
Hypoxic Stress ◽  
C6 Cells ◽  
Tumor Resistance ◽  
Glioblastoma Cells ◽  
Enzyme Expression ◽  
Hsp70 Chaperone ◽  
Therapeutic Tools ◽  
Enzyme Molecules

Hypoxia, which commonly accompanies tumor growth, depending on its strength may cause the enhancement of tumorigenicity of cancer cells or their death. One of the proteins targeted by hypoxia is glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and we demonstrated here that hypoxia mimicked by treating C6 rat glioblastoma cells with cobalt chloride caused an up-regulation of the enzyme expression, while further elevation of hypoxic stress caused the enzyme aggregation concomitantly with cell death. Reduction or elevation of GAPDH performed with the aid of specific shRNAs resulted in the augmentation of the tumorigenicity of C6 cells or their sensitization to hypoxic stress. Another hypoxia-regulated protein, Hsp70 chaperone, was shown to prevent the aggregation of oxidized GAPDH and to reduce hypoxia-mediated cell death. In order to release the enzyme molecules from the chaperone, we employed its inhibitor, derivative of colchicine. The compound was found to substantially increase aggregation of GAPDH and to sensitize C6 cells to hypoxia both in vitro and in animals bearing tumors with distinct levels of the enzyme expression. In conclusion, blocking the chaperonic activity of Hsp70 and its interaction with GAPDH may become a promising strategy to overcome tumor resistance to multiple environmental stresses and enhance existing therapeutic tools.

Oncogene-induced mitotic catastrophe and apoptosis in glioblastoma cells in vitro

2016 ◽  
Vol 4 ◽  
pp. 63-78
Author(s):  
Jolanta Zięba ◽  
Ewelina Stoczyńska-Fidelus ◽  
Piotr Rieske
Keyword(s):  
Cell Death ◽  
Cell Division ◽  
Mitotic Catastrophe ◽  
Glioblastoma Cells ◽  
Genetic Changes ◽  
Primary Brain Tumour ◽  
Problematic Issue ◽  
Vitro Analysis ◽  
In Vitro Analysis

Glioblastoma (GB) is one of the most prevalent and aggressive primary brain tumour. The median survival of GB patients who underwent standard therapy (neurosurgical resection, radiotherapy and chemotherapy) is 14 months. There for, GB treatment remains a great challenge. Furthermore, in vitro culturing of glioblastoma cells constitutes problematic issue. The reason for these difficulties might be found in 3 processes that were observed during in vitro cell culturing: apoptosis, mitotic catastrophe and senescence. The spontaneous occurrence of these phenomena leads to inhibition of cell division and / or cell death. Only a small percentage of glioblastoma cases present a genetic predisposition to be cultured in vitro. Analysis of available date indicate that 50% of GB cell lines are characterised by the coexistence of TP53 mutations and CDKN2A deletions. Comparing this percentage to 5% of coexistence of above-mentioned genetic changes in samples collected from patients (presenting) we can presume that the presence of those alterations has impact on stabilization of glioblastoma cell cultures. Importantly, proteins p16, p14 (encoded by CDKN2A) and TP53 protein are involved in the mentioned processes. In vitro dominates, senescence, apoptosis and mitotic catastrophe of glioblastoma cells. Identification of factors, responsible for spontaneous inhibition of cell division and / or cell death of glioblastoma cells in vitro, may provide a ground for a new GB therapeutic approach.

scholarly journals PACAP Modulates the Autophagy Process in an In Vitro Model of Amyotrophic Lateral Sclerosis

2020 ◽  
Vol 21 (8) ◽  
pp. 2943 ◽  
Author(s):  
Agata Grazia D’Amico ◽  
Grazia Maugeri ◽  
Salvatore Saccone ◽  
Concetta Federico ◽  
Sebastiano Cavallaro ◽  
...  
Keyword(s):  
Cell Death ◽  
Amyotrophic Lateral Sclerosis ◽  
Motor Neuron ◽  
Motor Neurons ◽  
In Vitro Model ◽  
Erk Signaling ◽  
Hypoxic Stress ◽  
Vitro Model ◽  
Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease of complex etiology leading to motor neuron degeneration. Many gene alterations cause this pathology, including mutation in Cu, Zn superoxide dismutase (SOD1), which leads to its gain of function. Mutant SOD1 proteins are prone to aberrant misfolding and create aggregates that impair autophagy. The hypoxic stress is strictly linked to the disease progression since it induces uncontrolled autophagy activation and the consequent high rates of cell death. Previously, we showed that pituitary adenylate cyclase-activating polypeptide (PACAP) exerts neurotrophic activity in cultured mSOD1 motor neurons exposed to serum deprivation. To date, no studies have examined whether the protective effect of PACAP on mSOD1 cells exposed to hypoxic insult is mediated through the regulation of the autophagy process. In the present study, we used the neuroblastoma-spinal cord-34 (NSC-34) cell line, stably expressing human wild type or mutant SOD1 G93A, to represent a well characterized in vitro model of a familial form of ALS. These cells were exposed to 100-µM desferrioxamine mesylate salt for 24h, to mimic the hypoxic stress affecting motor neurons during the disease progression. Our results showed that PACAP treatment significantly reduced cell death and hypoxia-induced mSOD1 accumulation by modulating the autophagy process in G93A motor neurons, as revealed by the decreased LC3II and the increased p62 levels, two autophagy indicators. These results were also confirmed by evaluating the vacuole formation detected through light chain 3 (LC3) immunofluorescence. Furthermore, the PACAP effects on autophagy seem to be mediated through the activation of the MAPK/ERK signaling pathway. Overall, our data demonstrated that PACAP exerts an ameliorative effect on the mSOD1 motor neuron viability by modulating a hypoxia-induced autophagy process through activation of MAPK/ERK signaling cascade.

Specific Cytostatic and Cytotoxic Effect of Dihydrochelerythrine in Glioblastoma Cells: Role of NF-κB/β-catenin and STAT3/IL-6 Pathways

2019 ◽  
Vol 18 (10) ◽  
pp. 1386-1393 ◽  
Author(s):  
Tereza C.C. Silva ◽  
Giselle P. de Faria Lopes ◽  
Noélio de J. Menezes-Filho ◽  
Diêgo M. de Oliveira ◽  
Ezequiel Pereira ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Viability ◽  
Annexin V ◽  
Experimental Models ◽  
Blue Dye ◽  
Dependent Manner ◽  
Glioblastoma Cells ◽  
U251 Cell ◽  
U251 Cells

Background: A glioblastoma is a primary CNS tumor that is more aggressive and lethal than other brain tumors. Its location, rapid proliferation, invasive growth, angiogenesis and immunosuppression are the main factors that limit its treatment, making it a major challenge to neuro-oncology. Objective: This study investigated the in vitro effects of the alkaloid dihydrochelerythrine (DHC), which is extracted from Zanthoxylum stelligerum, on the viability, proliferation, cell death and β-catenin, NFκB, STAT3/pSTAT3 and interleukins roles. Method: In vitro experimental models of human (U251 and GL-15) and murine (C6) glioblastoma cells were cultured in the presence of DHC at increasing concentrations for MTT assay and exclusion trypan blue dye to determine EC50. Afterward, C6 and U251 cells were treated with 100 µM DHC or DMSO 0.1% for cell cycle, annexin and expression of β-catenin/NFκB/STAT3/pSTAT3 by flow cytometry or immunofluorescence. Interleukin quantification was made by Cytometric Bead Array. Results: A significant decrease was observed in C6 and U251 cell viability in a time and dose-dependent manner. GL-15 cell viability decreased only when treated with 200 µM DHC. This maximum concentration affected neither astrocytes nor microglia viability. A cytostatic effect of DHC was observed in C6 and U251 cells after 48 h of 100 µM DHC treatment. After 72 h of DHC treatment, C6 presented 80% of annexin-V+ cells compared to 10% of annexin-V+ U251 cells. C6 cells demonstrated significant high levels of NFκ B and β-catenin cytoplasmic fraction. Additionally, DHC treatment resulted in higher significant levels of IL-6 than did other interleukins and STAT3 up-regulation in U251 cells. Conclusion: These results demonstrate that DHC acts as a chemosensitizing agent selective for glioma cells not affecting non-tumor cells. Considering tumor heterogeneity, DHC demonstrated an anti-cancer potential to activate different cell death pathways. DHC demonstrated could be used for chemotherapy and immunotherapy applications in glioblastomas in the future.

scholarly journals Molecular Mechanisms of Fenofibrate-Induced Metabolic Catastrophe and Glioblastoma Cell Death

2014 ◽  
Vol 35 (1) ◽  
pp. 182-198 ◽  
Author(s):  
Anna Wilk ◽  
Dorota Wyczechowska ◽  
Adriana Zapata ◽  
Matthew Dean ◽  
Jennifer Mullinax ◽  
...  
Keyword(s):  
Cell Death ◽  
Tumor Cell ◽  
Molecular Mechanisms ◽  
Prolonged Exposure ◽  
Lipid Lowering ◽  
Peroxisome Proliferator ◽  
Glioblastoma Cells ◽  
Tumor Cell Death ◽  
Metabolic Switch

Fenofibrate (FF) is a common lipid-lowering drug and a potent agonist of the peroxisome proliferator-activated receptor alpha (PPARα). FF and several other agonists of PPARα have interesting anticancer properties, and our recent studies demonstrate that FF is very effective against tumor cells of neuroectodermal origin. In spite of these promising anticancer effects, the molecular mechanism(s) of FF-induced tumor cell toxicity remains to be elucidated. Here we report a novel PPARα-independent mechanism explaining FF's cytotoxicityin vitroand in an intracranial mouse model of glioblastoma. The mechanism involves accumulation of FF in the mitochondrial fraction, followed by immediate impairment of mitochondrial respiration at the level of complex I of the electron transport chain. This mitochondrial action sensitizes tested glioblastoma cells to the PPARα-dependent metabolic switch from glycolysis to fatty acid β-oxidation. As a consequence, prolonged exposure to FF depletes intracellular ATP, activates the AMP-activated protein kinase–mammalian target of rapamycin–autophagy pathway, and results in extensive tumor cell death. Interestingly, autophagy activators attenuate and autophagy inhibitors enhance FF-induced glioblastoma cytotoxicity. Our results explain the molecular basis of FF-induced glioblastoma cytotoxicity and reveal a new supplemental therapeutic approach in which intracranial infusion of FF could selectively trigger metabolic catastrophe in glioblastoma cells.

scholarly journals EXTH-44. INHIBITION OF MerTK ACTIVATES GLIOBLASTOMA-ASSOCIATED MACROPHAGES AND INDUCES TUMOR CELL DEATH IN GLIOMA MICROENVIRONMENT

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi91-vi91
Author(s):  
Yu-Ting Su ◽  
Madison Butler ◽  
Lee Hwang ◽  
Dragan Maric ◽  
Shelton Earp ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Growth ◽  
Tumor Microenvironment ◽  
Tumor Cell ◽  
Colony Formation ◽  
Glioblastoma Cells ◽  
Tumor Cell Death ◽  
Glioma Microenvironment

Abstract BACKGROUND Glioblastoma-associated macrophages and microglia (GAMs) are the predominant immune cells in the tumor microenvironment. Activation of MerTK, a receptor tyrosine kinase, triggers efferocytosis and polarizes GAMs to an immunosuppressive phenotype, promoting glioma growth. Our previous findings showed that UNC2371, a small-molecule inhibitor of MerTK, induced a less immunosuppressive phenotype of GAMs. Here, we investigate the role of MerTK inhibition on glioblastoma cells in the tumor microenvironment in vitro and in vivo. METHODS Cytotoxicity of UNC2371 in glioblastoma cells was determined by cell viability and colony formation assays. The protein expression of MerTK, AKT, and Erk were quantified by Western blotting in UNC2371-treated glioblastoma cells. A syngeneic GL261 mouse orthotopic glioblastoma model was used to evaluate the survival benefit of UNC2371 treatment. Fluorescent multiplex immunohistochemistry (IHC) was used to evaluate the expression of CD206, an anti-inflammatory marker on GAMs in murine brain tumor tissues. RESULTS UNC2371 inhibited GBM cell growth with an EC50 < 100 nM in both human U251 and mouse GL261 glioma cells, but not in GAMs. UNC2371-induced cell death and decreased cell proliferation were demonstrated by colony formation assays. UNC2371 decreased protein expression of phosphorylated MerTK, AKT, and Erk, which are essential for cell survival signaling, in U251 and GL261 cells. Furthermore, UNC2371 treatment prolonged survival in the mouse orthotopic GL261 glioblastoma model, suggesting that UNC2371 induces glioma cell death. A decreased of CD206+ GAMs was found in mice glioma tissues by fluorescent multiplex IHC, consistent with our previous findings in the in vitro cell-based assays. These data suggest that in addition to alleviate immunosuppression in the glioma microenvironment, UNC2371 directly inhibits GBM cell growth in vitro and in vivo. CONCLUSION Our findings suggest that UNC2371 has a therapeutic benefit via promoting GAM polarization towards proinflammatory status in the glioblastoma microenvironment and unexpectedly, inducing tumor cell death.

scholarly journals In vitro Antineoplastic Activity of Dye Compounds on Human Glioblastoma Cells

2019 ◽  
Vol 70 (1) ◽  
pp. 112-117
Author(s):  
Oana Alexandru ◽  
Ada Maria Georgescu ◽  
Alexandra Dragoi ◽  
Marius Eugen Ciurea ◽  
Citto Iulian Taisescu ◽  
...  
Keyword(s):  
Cell Death ◽  
Apoptotic Cell ◽  
Malignant Gliomas ◽  
Drug Efficacy ◽  
High Grade Glioma ◽  
Glioblastoma Cells ◽  
Incurable Disease ◽  
Apoptotic Effect ◽  
Low Passage

Dyes are an important class of natural and synthetic compounds, recently studied as potential anticancer drugs. Among various natural dye molecules, Curcumin was extensively studied in treatment of malignant gliomas, a highly incurable disease. Curcumin was reported to induce cell death in malignant gliomas by induction autophagy and apoptosis. We have previously reported that Helianthin, a synthetic dye compound, also induced apoptotic cell death in high grade glioma cells. In this study we evaluated the antiproliferative and the apoptotic effect of Curcumin and Helianthin on a human low passage glioblastoma cell line. We found that both compounds displayed antiproliferative properties on glioblastoma cells, however, at equimolar concentrations, Helianthin induced more cytotoxic effect than Curcumin. IC50 value is considered a good indicator of drug efficacy. We found that Helianthin required a lower concentration to achieve IC50 (16.9.735 �14.8 mM) than Curcumin (68.5 �12.3 mM). We also found that Curcumin and Helianthin treatment induced caspase 3, 8 and 9 activation in glioblastoma cells. This study may lead to a widespread search for dye agents that may represent an untapped source of drugs for cancer treatment.

Enhanced expression of proapoptotic and autophagic proteins involved in the cell death of glioblastoma induced by synthetic glycans

2014 ◽  
Vol 120 (6) ◽  
pp. 1298-1308 ◽  
Author(s):  
Ahmad Faried ◽  
Muhammad Zafrullah Arifin ◽  
Shogo Ishiuchi ◽  
Hiroyuki Kuwano ◽  
Shin Yazawa
Keyword(s):  
Cell Death ◽  
Mouse Model ◽  
Anticancer Agents ◽  
Anticancer Agent ◽  
Adenosine Diphosphate ◽  
Glioblastoma Cells ◽  
Enhanced Expression ◽  
Autophagic Proteins

Object Glioblastoma is the most aggressive malignant brain tumor, and overall patient survival has not been prolonged even by conventional therapies. Previously, the authors found that chemically synthesized glycans could be anticancer agents against growth of a series of cancer cells. In this study, the authors examined the effects of glycans on the growth of glioblastoma cells both in vitro and in vivo. Methods The authors investigated not only the occurrence of changes in the cell signaling molecules and expression levels of various proteins related to cell death, but also a mouse model involving the injection of glioblastoma cells following the administration of synthetic glycans. Results Synthetic glycans inhibited the growth of glioblastoma cells, induced the apoptosis of the cells with cleaved poly (adenosine diphosphate-ribose) polymerase (PARP) expression and DNA fragmentation, and also caused autophagy, as shown by the detection of autophagosome proteins and monodansylcadaverine staining. Furthermore, tumor growth in the in vivo mouse model was significantly inhibited. A dramatic induction of programmed cell death was found in glioblastoma cells after treatment with synthetic glycans. Conclusions These results suggest that synthetic glycans could be a promising novel anticancer agent for performing chemotherapy against glioblastoma.

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