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

Cover Page


Cite item

Full Text

Open Access Open Access
Restricted Access Access granted
Restricted Access Subscription or Fee Access

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.

Full Text

Restricted Access

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

References

  1. Miller KD, Ostrom QT, Kruchko C, et al. Brain and other central nervous system tumor statistics. CA Cancer J Clin. 2021;71(5):381–406. doi: 10.3322/caac.21693
  2. Skaga E, Kulesskiy E, Fayzullin A, et al. Intertumoral heterogeneity in patient-specific drug sensitivities in treatment-naïve glioblastoma. BMC Cancer. 2019;19(1):628. doi: 10.1186/s12885-019-5861-4
  3. Taylor OG, Brzozowski JS, Skelding KA. Glioblastoma multiforme: an overview of emerging therapeutic targets. Front Oncol. 2019;9:963. doi: 10.3389/fonc.2019.00963
  4. Chernov AN, Alaverdian DA, Galimova ES, et al. The phenomenon of multidrug resistance in glioblastomas. Hematol Oncol Stem Cell Ther. 2022;15(2):1–7. doi: 10.1016/j.hemonc.2021.05.006
  5. Olivier C, Oliver L, Lalier L, et al. Drug resistance in glioblastoma: The two faces of oxidative stress. Front Mol Biosci. 2021;7:620677. doi: 10.3389/fmolb.2020.620677
  6. Marzagalli M, Fontana F, Raimondi M, et al. Cancer stem cells-key players in tumor relapse. Cancers (Basel). 2021;13(3):376. doi: 10.3390/cancers13030376
  7. Bazzoni R, Bentivegna A. Role of notch signaling pathway in glioblastoma multiforme pathogenesis. Cancers (Basel). 2019;11(3):292. doi: 10.3390/cancers11030292
  8. Bhuvanalakshmi G, Gamit N, Patil M, et al. Stemness, pluripotentiality, and wnt antagonism: sFRP4, a wnt antagonist mediates pluripotency and stemness in glioblastoma. Cancers (Basel). 2018;11(1):25. doi: 10.3390/cancers11010025
  9. Dymova MA, Kuligina EV, Richter VA. Molecular mechanisms of drug resistance in glioblastoma. Int J Mol Sci. 2021;22(12):6385. doi: 10.3390/ijms22126385
  10. Diehn M, Cho RW, Lobo NA, et al. Association of reactive oxygen species levels and radioresistance in cancer stem cells. Nature. 2009;458(7239):780–783. doi: 10.1038/nature07733
  11. Wang L, Zhang X, Cui G, et al. A novel agent exerts antitumor activity in breast cancer cells by targeting mitochondrial complex II. Oncotarget. 2016;7(22):32054–32064. doi: 10.18632/oncotarget.8410
  12. Redza-Dutordoir M, Averill-Bates DA. Activation of apoptosis signalling pathways by reactive oxygen species. Biochim Biophys Acta. 2016;1863(12):2977–2992. doi: 10.1016/j.bbamcr.2016.09.012
  13. Li Y, Chang Y, Ye N, et al. Advanced glycation end products-induced mitochondrial energy metabolism dysfunction alters proliferation of human umbilical vein endothelial cells. Mol Med Rep. 2017;15(5):2673–2680. doi: 10.3892/mmr.2017.6314
  14. D’Alterio C, Scala S, Sozzi G, et al. Paradoxical effects of chemotherapy on tumor relapse and metastasis promotion. Semin Cancer Biol. 2020;60:351–361. doi: 10.1016/j.semcancer.2019.08.019
  15. Franco ML, Nadezhdin KD, Light TP, et al. Interaction between the transmembrane domains of neurotrophin receptors p75 and TrkA mediates their reciprocal activation . J Biol Chem. 2021;297(2):100926. doi: 10.1016/j.jbc.2021.100926
  16. Huang EJ, Reichardt LF. Neurotrophins: Roles in neuronal development and function. Annu Rev Neurosci. 2001;24:677–736. doi: 10.1146/annurev.neuro.24.1.677
  17. Donato MD, Galasso G, Giovannelli P, et al. Targeting the nerve growth factor signaling impairs the proliferative and migratory phenotype of triple-negative breast cancer cells. Front Cell Dev Biol. 2021;9:676568. doi: 10.3389/fcell.2021.676568
  18. Paul AB, Grant ESF, Habib K. The expression and localisation of beta-nerve growth factor (beta-NGF) in benign and malignant human prostate tissue: relationship to neuroendocrine differentiation. Br J Cancer. 1996;74(12):1990–1996. doi: 10.1038/bjc.1996.665
  19. Sierra-Fonseca JA, Najera O, Martinez-Jurado J, et al. Nerve growth factor induces neurite outgrowth of PC12 cells by promoting Gβγ-microtubule interaction. BMC Neurosci. 2014;15:132. doi: 10.1186/s12868-014-0132-4
  20. Li R, Li D, Wu C, et al. Nerve growth factor activates autophagy in Schwann cells to enhance myelin debris clearance and to expedite nerve regeneration. Theranostics. 2020;10(4):1649–1677. doi: 10.7150/thno.40919
  21. Buravlev VM, Veprintsev BN, Viktorov IV, et al. Guide to the cultivation of nervous tissue. Methods. Technique. Problems. Moscow: Nauka; 1988. 315 p. (In Russ.) EDN: VTGDTL
  22. Li C, Zhou C, Wang S, et al. Sensitization of glioma cells to tamoxifen-induced apoptosis by Pl3-kinase inhibitor through the GSK-3β/β-catenin signaling pathway. PLoS One. 2011;6(10):e27053. doi: 10.1371/journal.pone.0027053
  23. Ge H, Mu L, Jin L, et al. Tumor associated CD70 expression is involved in promoting tumor migration and macrophage infiltration in GBM. Int J Cancer. 2017;141(7):1434–1444. doi: 10.1002/ijc.30830
  24. Gu X, Ma Y, Liu Y, et al. Measurement of mitochondrial respiration in adherent cells by seahorse XF96 cell Mito stress. STAR Protoc. 2020;2(1):100245. doi: 10.1016/j.xpro.2020.100245
  25. Little AC, Kovalenko I, Goo LE, et al. High-content fluorescence imaging with the metabolic flux assay reveals insights into mitochondrial properties and functions. Commun Biol. 2020;3(1):271. doi: 10.1038/s42003-020-0988-z
  26. Li Y, Chang Y, Ye N, et al. Advanced glycation end products-induced mitochondrial energy metabolism dysfunction alters proliferation of human umbilical vein endothelial cells. Mol Med Rep . 2017;15(5):2673–2680. doi: 10.3892/mmr.2017.6314
  27. Agilent Seahorse XF Glycolysis Stress Test Kit User Guide Kit 103020-100. USA: Agilent Technologies, Inc.; 2019. 22 p.
  28. Glantz SA. Primer of Biostatistics. 4th ed. McGraw-Hill, Health Professions Division; 1997.
  29. Zhou X, Hao Q, Liao P, et al. Nerve growth factor receptor negates the tumor suppressor p53 as a feedback regulator. eLife. 2016;5:e15099. doi: 10.7554/eLife.1509
  30. Aubert L, Guilbert M, Corbet C, et al. NGF-induced TrkA/CD44 association is involved in tumor aggressiveness and resistance to lestaurtinib. Oncotarget. 2015;6(12):9807–9819. doi: 10.18632/oncotarget.3227
  31. Johnston AL, Lun X, Rahn JJ, et al. The p75 neurotrophin receptor is a central regulator of glioma invasion. PLoS Biol. 2007;5(8):e212. doi: 10.1371/journal.pbio.0050212
  32. Tong B, Pantazopoulou V, Johansson E, et al. The p75 neurotrophin receptor enhances HIF-dependent signaling in glioma. Exp Cell Res. 2018;371(1):122–129. doi: 10.1016/j.yexcr.2018.08.002
  33. Wang TC, Luo SJ, Lin CL, et al. Modulation of p75 neurotrophin receptor under hypoxic conditions induces migration and invasion of C6 glioma cells. Clin Exp Metastasis. 2015;32(1):73–81. doi: 10.1007/s10585-014-9692-z
  34. Donato MD, Galasso G, Giovannelli P, et al. Targeting the nerve growth factor signaling impairs the proliferative and migratory phenotype of triple-negative breast cancer cells. Front Cell Dev Biol. 2021;9:676568. doi: 10.3389/fcell.2021.676568
  35. Arthurs AL, Keating DJ, Stringer BW, Conn SJ. The suitability of glioblastoma cell lines as models for primary glioblastoma cell metabolism. Cancers (Basel). 2020;12(12):3722. doi: 10.3390/cancers12123722
  36. Warburg O. On the origin of cancer cells. Science. 1956;123:309–314 . doi: 10.1126/science.123.3191.309
  37. Vlashi E, Lagadec C, Vergnes L, et al. Metabolic state of glioma stem cells and nontumorigenic cells. Proc Natl Acad Sci USA. 2011;108(38):16062–16067. doi: 10.1073/pnas.1106704108
  38. Di K, Lomeli N, Bota DA, et al. Magmas inhibition as a potential treatment strategy in malignant glioma. J Neurooncol. 2019;141(2):267–276. doi: 10.1007/s11060-018-03040-8
  39. Kanu LN, Ross AE, Farhat W, et al. Development and characterization of a photocrosslinkable, chitosan-based, nerve growth factor-eluting hydrogel for the ocular surface. Transl Vis Sci Technol. 2024;13(6):12. doi: 10.1167/tvst.13.6.12
  40. Nerve Growth Factor Eye Drops Treatment in Patients with Retinitis Pigmentosa and Cystoid Macular Edema (NEMO). Available from: https://ctv.veeva.com/study/nerve-growth-factor-eye-drops-treatment-in-patients-with-retinitis-pigmentosa-and-cystoid-macular-ed . Accessed: 29 June 2024.
  41. An 8-week Study to Evaluate Safety and Efficacy of NGF Eye Drops Solution Versus Vehicle in Patients with Dry Eye. Available from: https://clinicaltrials.gov/study/NCT03019627 . Accessed: 29 June 2024.
  42. Cvrljevic AN, Akhavan D, Wu M, et al. Activation of Src induces mitochondrial localization of de2-7EGFR (EGFRvIII) in glioma cells: implications for glucose metabolism. J Cell Sci. 2011;124(Pt 17):2938–2950 . doi: 10.1242/jcs.083295

Supplementary files

Supplementary Files
Action
1. JATS XML
Download
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

Download (148KB)
Indexing metadata
3. Fig. 2. Experimental design for assessing glycolytic capacity of cells using the Seahorse Bioscience XF24 analyzer

Download (119KB)
Indexing metadata
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

Download (288KB)
Indexing metadata
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

Download (306KB)
Indexing metadata
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

Download (161KB)
Indexing metadata
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

Download (182KB)
Indexing metadata

Copyright (c) 2024 Eco-Vector


СМИ зарегистрировано Федеральной службой по надзору в сфере связи, информационных технологий и массовых коммуникаций (Роскомнадзор).
Регистрационный номер и дата принятия решения о регистрации СМИ: серия ПИ № ФС 77 - 74760 от 29.12.2018 г.