Nerve growth factor: impact on migration, clonogenicity, and bioenergetic metabolism of mitochondria of glioma U251 cells

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Abstract

BACKGROUND: Glioblastoma is the most malignant tumor of the central nervous system. Temozolomide is the standard treatment for gliomas, and its use often leads to drug resistance and relapse of glioblastoma. Therefore, further research is needed to find other drugs that can improve the effectiveness of standard treatments.

AIM: The goal is to study the effects of nerve growth factor, temozolomide on clonogenicity, migration and energy metabolism of mitochondria of human U251 glioma cells.

MATERIALS AND METHODS: The study was conducted on human U251 glioma cells. A colony formation test was used to evaluate the ability of glioma cells to form colonies in vitro. Migration of U251 glioma cells was assessed by the Scratch Assay. To study mitochondrial metabolism in glioma cells, oxygen consumption rate and extracellular acidification rate were measured using the Seahorse XF CellMito and Seahorse XF Glycolysis Stress Test kits, respectively.

RESULTS: We found that nerve growth factor (7.55 × 10–3 µM) and temozolomide (155 µM) inhibited the clonogenicity of U251 glioma cells by 66.2% and 73.5–81.3% within 1–2 days, respectively. Exposure to nerve growth factor (7.55 × 10–3 µM) also suppresses U251 glioma cell migration on days 3 and 4. Temozolomide (155 µM) inhibits glioma cell migration on days 1–3. The anti-clonogenic and anti-migratory activities of nerve growth factor and temozolomide may be associated with their ability to reduce the basal rate of oxygen consumption, inhibit adenosine triphosphate synthetase and maximum mitochondrial respiration in human U251 glioma cells. Nerve growth factor and temozolomide did not affect glycolysis, glycolytic capacity, and glycolytic reserve in U251 glioma cells compared to controls.

CONCLUSIONS: Thus, nerve growth factor and temozolomide inhibit migration, clonogenicity, and bioenergetic metabolism of mitochondria in U251 glioma cells, exhibiting anti-mitogenic, anti-migration, and reducing energy metabolism effects.

About the authors

Alexandr N. Chernov

Institute of Experimental Medicine; Saint Petersburg State Pediatric Medical University

Author for correspondence.
Email: al.chernov@mail.ru
ORCID iD: 0000-0003-2464-7370
SPIN-code: 8454-1568

Cand. Sci. (Biology), Senior Research Associate, Department of General Pathology and Pathological Physiology, Assistant of the Department of Biological Chemistry

Russian Federation, 12 Academician Pavlov St., Saint Petersburg, 197022; Saint Petersburg

Ruslan I. Glushakov

Saint Petersburg State Pediatric Medical University

Email: glushakovruslan1@gmail.com
ORCID iD: 0000-0002-0161-5977
SPIN-code: 6860-8990

Dr. Sci. (Medicine), Assistant Professor of the Department of Pharmacology with a course in Clinical Pharmacology and Pharmacoeconomics

Russian Federation, Saint Petersburg

Sofia S. Landynya

Institute of Experimental Medicine; Saint Petersburg State University

Email: sofia.landynya@mail.ru

Laboratory assistant of the Department of General Pathology and Pathophysiology, laboratory assistant of the Department of Biochemistry

Russian Federation, 12 Academician Pavlov St., Saint Petersburg, 197022; Saint Petersburg

Yaroslav A. Sharapov

Institute of Experimental Medicine; Saint Petersburg State University

Email: yarostloff@yandex.ru

Laboratory assistant of the Department of General Pathology and Pathophysiology, laboratory assistant of the Department of Biochemistry

Russian Federation, 12 Academician Pavlov St., Saint Petersburg, 197022; Saint Petersburg

Elvira S. Galimova

Institute of Experimental Medicine; Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences

Email: elya-4@yandex.ru
ORCID iD: 0000-0002-8773-0932
SPIN-code: 1989-2143

Cand. Sci. (Biology), Senior Research Associate, Department of General Pathology and Pathological Physiology, Senior Research Associate, Interdisciplinary Laboratory of Neurobiology

Russian Federation, 12 Academician Pavlov St., Saint Petersburg, 197022; Saint Petersburg

Olga V. Shamova

Institute of Experimental Medicine; Saint Petersburg State University

Email: oshamova@yandex.ru
ORCID iD: 0000-0002-5168-2801
SPIN-code: 2913-4726

Assistant Professor, Corresponding Member of the RAS, Head of the Department of General Pathology and Pathophysiology, Head of the Department of Biochemistry, Professor of the Department

Russian Federation, 12 Academician Pavlov St., Saint Petersburg, 197022; Saint Petersburg

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Supplementary files

Supplementary Files
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2. Fig. 1. Study of different types of mitochondrial respiration by oxygen uptake rate using the XF24 Seahorse Bioscience analyzer. ATP, adenosine triphosphate; FCCP, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone

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3. Fig. 2. Experimental design for assessing glycolytic capacity of cells using the Seahorse Bioscience XF24 analyzer

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4. Fig. 3. Microscopic images showing the process of colony formation by human glioma U251 cells in the presence of nerve growth factor and temozolomide. Clonogenic capacity of human glioma U251 cells on days 1–3 in the control ( a , b , c ), after 1–3 days of exposure to nerve growth factor (7.55 × 10 –3 μmol/l) ( d , e , f ) and temozolomide (155 μmol/l) ( g , h , i ). Objective magnification ×10. Scale bar 400 μm

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5. Fig. 4. Microscopic images showing the migration of U251 glioma cells into the “scratch” zone: in the control for 1–3 days ( a , b , c ), after 1–3 days of exposure to nerve growth factor (7.55 10–3 μmol/l) ( d , e , f ) and temozolomide (155 μmol/l) ( g , h , i ). Total magnification ×100. Scale bar 200 μm

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6. Fig. 5. Oxygen consumption rate of human U251 glioma cells exposed to nerve growth factor (NGF) and temozolomide (TMZ) compared to control. ** p < 0.01, *** p < 0.001, **** p < 0.0001

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7. Fig. 6. The rate of extracellular acidification of the environment in U251 glioma cells under the influence of nerve growth factor (NGF) (7.55 × 10 –3 μmol/l) and temozolomide (TMZ) (155 μmol/l) compared to the control. *** p < 0.001

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