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

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.

Keywords

human glioma U251 / nerve growth factor / temozolomide / migration / clonogenicity / energy metabolism of mitochondria / oxygen consumption rate / extracellular acidification rate

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] Miller K.D., Ostrom Q.T., Kruchko C., et al. Brain and other central nervous system tumor statistics // CA Cancer J Clin. 2021. Vol. 71, N 5. P. 381–406. doi: 10.3322/caac.21693

[3] 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

[4] Skaga E., Kulesskiy E., Fayzullin A., et al. Intertumoral heterogeneity in patient-specific drug sensitivities in treatment-naïve glioblastoma // BMC Cancer. 2019. Vol. 19, N 1. P. 628. doi: 10.1186/s12885-019-5861-4

[5] 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

[6] Taylor O.G., Brzozowski J.S., Skelding K.A. Glioblastoma multiforme: an overview of emerging therapeutic targets // Front Oncol. 2019. Vol. 9. P. 963. doi: 10.3389/fonc.2019.00963

[7] 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

[8] Chernov A.N., Alaverdian D.A., Galimova E.S., et al. The phenomenon of multidrug resistance in glioblastomas // Hematol Oncol Stem Cell Ther. 2022. Vol. 15, N 2. P. 1–7. doi: 10.1016/j.hemonc.2021.05.006

[9] 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

[10] Olivier C., Oliver L., Lalier L., et al. Drug resistance in glioblastoma: The two faces of oxidative stress // Front Mol Biosci. 2021. Vol. 7. P. 620677. doi: 10.3389/fmolb.2020.620677

[11] 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

[12] Marzagalli M., Fontana F., Raimondi M., et al. Cancer stem cells-key players in tumor relapse // Cancers (Basel). 2021. Vol. 13, N 3. P. 376. doi: 10.3390/cancers13030376

[13] Bazzoni R, Bentivegna A. Role of notch signaling pathway in glioblastoma multiforme pathogenesis. Cancers (Basel). 2019;11(3):292. doi: 10.3390/cancers11030292

[14] Bazzoni R., Bentivegna A. Role of notch signaling pathway in glioblastoma multiforme pathogenesis // Cancers (Basel). 2019. Vol. 11, N 3. P. 292. doi: 10.3390/cancers11030292

[15] 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

[16] 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. Vol. 11, N 1. P. 25. doi: 10.3390/cancers11010025

[17] 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

[18] Dymova M.A., Kuligina E.V., Richter V.A. Molecular mechanisms of drug resistance in glioblastoma // Int J Mol Sci. 2021. Vol. 22, N 12. P. 6385. doi: 10.3390/ijms22126385

[19] 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

[20] Diehn M., Cho R.W., Lobo N.A., et al. Association of reactive oxygen species levels and radioresistance in cancer stem cells // Nature. 2009. Vol. 458, N 7239. P. 780–783. doi: 10.1038/nature07733

[21] 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

[22] 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. Vol. 7, N 22. P. 32054–32064. doi: 10.18632/oncotarget.8410

[23] 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

[24] Redza-Dutordoir M., Averill-Bates D.A. Activation of apoptosis signalling pathways by reactive oxygen species // Biochim Biophys Acta. 2016. Vol. 1863, N 12. P. 2977–2992. doi: 10.1016/j.bbamcr.2016.09.012

[25] 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

[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. Vol. 15, N 5. P. 2673–2680. doi: 10.3892/mmr.2017.6314

[27] 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

[28] D’Alterio C., Scala S., Sozzi G., et al. Paradoxical effects of chemotherapy on tumor relapse and metastasis promotion // Semin Cancer Biol. 2020. Vol. 60. P. 351–361. doi: 10.1016/j.semcancer.2019.08.019

[29] 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

[30] Franco M.L., Nadezhdin K.D., Light T.P., et al. Interaction between the transmembrane domains of neurotrophin receptors p75 and TrkA mediates their reciprocal activation // J Biol Chem. 2021. Vol. 297, N 2. P. 100926. doi: 10.1016/j.jbc.2021.100926

[31] 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

[32] Huang E.J., Reichardt L.F. Neurotrophins: Roles in neuronal development and function // Annu Rev Neurosci. 2001. Vol. 24. P. 677–736. doi: 10.1146/annurev.neuro.24.1.677

[33] 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

[34] Donato M.D., 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. Vol. 9. P. 676568. doi: 10.3389/fcell.2021.676568

[35] 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

[36] Paul A.B., Grant E.S.F., 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. Vol. 74, N 12. P. 1990–1996. doi: 10.1038/bjc.1996.665

[37] 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

[38] Sierra-Fonseca J.A., Najera O., Martinez-Jurado J., et al. Nerve growth factor induces neurite outgrowth of PC12 cells by promoting Gβγ-microtubule interaction // BMC Neurosci. 2014. Vol. 15. P. 132. doi: 10.1186/s12868-014-0132-4

[39] 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

[40] 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. Vol. 10, N 4. P. 1649–1677. doi: 10.7150/thno.40919

[41] 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

[42] Буравлев В.М., Вепринцев Б.Н., Викторов И.В. и др. Руководство по культивированию нервной ткани. Методы. Техника. Проблемы. Москва: Наука, 1988. 315 с. EDN: VTGDTL

[43] 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

[44] 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. Vol. 6, N 10. P. e27053. doi: 10.1371/journal.pone.0027053

[45] 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

[46] 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. Vol. 141, N 7. P. 1434–1444. doi: 10.1002/ijc.30830

[47] 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

[48] Gu X., Ma Y., Liu Y., et al. Measurement of mitochondrial respiration in adherent cells by seahorse XF96 cell mito stress // STAR Protoc. 2020. Vol. 2, N 1. P. 100245. doi: 10.1016/j.xpro.2020.100245

[49] 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

[50] Little A.C., Kovalenko I., Goo L.E., et al. High-content fluorescence imaging with the metabolic flux assay reveals insights into mitochondrial properties and functions // Commun Biol. 2020. Vol. 3, N 1. P. 271. doi: 10.1038/s42003-020-0988-z

[51] 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

[52] 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. Vol. 15, N 5. P. 2673–2680. doi: 10.3892/mmr.2017.6314

[53] Agilent Seahorse XF Glycolysis Stress Test Kit User Guide Kit 103020-100. USA: Agilent Technologies, Inc.; 2019. 22 p.

[54] Agilent Seahorse XF Glycolysis Stress Test Kit User Guide Kit 103020-100. USA: Agilent Technologies, Inc., 2019. 22 p.

[55] Glantz SA. Primer of Biostatistics. 4th ed. McGraw-Hill, Health Professions Division; 1997.

[56] Гланц С. Медико-биологическая статистика. Москва: Практика, 1999. 459 с.

[57] 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

[58] Zhou X., Hao Q., Liao P., et al. Nerve growth factor receptor negates the tumor suppressor p53 as a feedback regulator // eLife. 2016. Vol. 5. P. e15099. doi: 10.7554/eLife.1509

[59] 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

[60] Aubert L., Guilbert M., Corbet C., et al. NGF-induced TrkA/CD44 association is involved in tumor aggressiveness and resistance to lestaurtinib // Oncotarget. 2015. Vol. 6, N 12. P. 9807–9819. doi: 10.18632/oncotarget.3227

[61] 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

[62] Johnston A.L., Lun X., Rahn J.J., et al. The p75 neurotrophin receptor is a central regulator of glioma invasion // PLoS Biol. 2007. Vol. 5, N 8. P. e212. doi: 10.1371/journal.pbio.0050212

[63] 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

[64] Tong B., Pantazopoulou V., Johansson E., et al. The p75 neurotrophin receptor enhances HIF-dependent signaling in glioma // Exp Cell Res. 2018. Vol. 371, N 1. P. 122–129. doi: 10.1016/j.yexcr.2018.08.002

[65] 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

[66] Wang T.C., Luo S.J., Lin C.L., et al. Modulation of p75 neurotrophin receptor under hypoxic conditions induces migration and invasion of C6 glioma cells // Clin Exp Metastasis. 2015. Vol. 32, N 1. P. 73–81. doi: 10.1007/s10585-014-9692-z

[67] 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

[68] Donato M.D., 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. Vol. 9. P. 676568. doi: 10.3389/fcell.2021.676568

[69] 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

[70] Arthurs A.L., Keating D.J., Stringer B.W., Conn S.J. The suitability of glioblastoma cell lines as models for primary glioblastoma cell metabolism // Cancers (Basel). 2020. Vol. 12, N 12. P. 3722. doi: 10.3390/cancers12123722

[71] Warburg O. On the origin of cancer cells. Science. 1956;123:309–314 . doi: 10.1126/science.123.3191.309

[72] Warburg O. On the origin of cancer cells // Science. 1956. Vol. 123. P. 309–314. doi: 10.1126/science.123.3191.309

[73] 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

[74] Vlashi E., Lagadec C., Vergnes L., et al. Metabolic state of glioma stem cells and nontumorigenic cells // Proc Natl Acad Sci USA. 2011. Vol. 108, N 38. P. 16062–16067. doi: 10.1073/pnas.1106704108

[75] 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

[76] Di K., Lomeli N., Bota D.A., et al. Magmas inhibition as a potential treatment strategy in malignant glioma // J Neurooncol. 2019. Vol. 141, N 2. P. 267–276. doi: 10.1007/s11060-018-03040-8

[77] 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

[78] Kanu L.N., Ross A.E., 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. Vol. 13, N 6. P. 12. doi: 10.1167/tvst.13.6.12

[79] 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.

[80] Nerve Growth Factor Eye Drops Treatment in Patients with Retinitis Pigmentosa and Cystoid Macular Edema (NEMO). Режим доступа: https://ctv.veeva.com/study/nerve-growth-factor-eye-drops-treatment-in-patients-with-retinitis-pigmentosa-and-cystoid-macular-ed . Дата обращения: 29.06.2024.

[81] 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.

[82] An 8-week Study to Evaluate Safety and Efficacy of NGF Eye Drops Solution Versus Vehicle in Patients with Dry Eye. Режим доступа: https://clinicaltrials.gov/study/NCT03019627 . Дата обращения: 29.06.2024.

[83] 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

[84] Cvrljevic A.N., 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. Vol. 124, N Pt 17. P. 2938–2950. doi: 10.1242/jcs.083295

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