Neurochemical Research

, Volume 41, Issue 4, pp 844–854 | Cite as

Neuroprotective Effects of Etidronate and 2,3,3-Trisphosphonate Against Glutamate-Induced Toxicity in PC12 Cells

  • Wen Li
  • Yuen-Ki Cheong
  • Hui Wang
  • Guogang Ren
  • Zhuo YangEmail author
Original Paper


Etidronate is one of the best known bisphosphonates (BP) derivatives. It is often used as a reference drug in research related to hypercalcaemia and other common bone diseases. 2,3,3-trisphosphonate (TrisPP) is brand new analogue of BP, that also contains a ‘germinal bisphosphonate’ unit with an additional phosphoryl group attached in proximity to the BP unit. It is known that BPs bind to calcium by chemisorptions to form Ca-BP complexes through (O)P–C–P(O) moiety and hydrogen coordinations, and so they suppress calcium flow by interfering with Ca2+ channel operations. The mechanistic actions of BP, involving interactions and regulations of Ca2+, are somewhat similar to the pathogenesis of well-known neurodegenerative disorders, such as Alzheimer’s disease, Parkinson’s disease and Huntington’s disease. To investigate if neuroprotective effects are exhibited by the compounds of interests, we used a rat adrenal pheochromocytoma cell line (PC12) as our in vitro model to observe any occurrence of neuron inter-reflection. We pre-treated these PC12 cells with etidronate and TrisPP before challenging the cells with a high concentration of the neurotoxin, glutamate. Our data showed that pre-treatment with 100 μM etidronate partially ameliorated the glutamate-induced decrease in cell viability (47 %), whereas pre-treating cells with 10–100 μM TrisPP showed remarkable cell protection (78–86 %). Moreover, pre-treatments of the cells with etidronate or TrisPP attenuated cell apoptosis, reactive oxygen species generation, Ca2+ overloading and caspase-3 protein expression, which were associated with a remarkable increase in superoxide dismutase activity in our glutamate-injured PC12 cells. Therefore, this study supports the notion that etidronate and TrisPP may be promising neuroprotective agents.


Etidronate 2,3,3-Trisphosphonate PC12 cells Glutamate Neuroprotection 



This work was supported by grant from Tianjin Research Program of Application Foundation and Advanced Technology (14JCZDJC35000), the National Natural Science Foundation of China (81571804) and the UK Royal Academy of Engineering (RAEng. 1213RECI052).


  1. 1.
    Fulfaro F, Casuccio A, Ticozzi C, Ripamonti C (1998) The role of bisphosphonates in the treatment of painful metastatic bone disease: a review of phase III trials. Pain 78:157–169CrossRefPubMedGoogle Scholar
  2. 2.
    Fleisch H (1998) Bisphosphonates: mechanisms of action. Endocr Rev 19:80–100CrossRefPubMedGoogle Scholar
  3. 3.
    Russell RG (2011) Bisphosphonates: the first 40 years. Bone 49:2–19CrossRefPubMedGoogle Scholar
  4. 4.
    Russell RGG, Xia Z, Dunford JE, Oppermann U, Kwaasi A, Hulley PA, Kavanagh KL, Triffitt JT, Lundy MW, Phipps RJ, Barnett BL, Coxon FP, Rogers MJ, Watts NB, Ebetino FH (2007) Bisphosphonates: an update on mechanisms of action and how these relate to clinical efficacy. Ann N Y Acad Sci 1117:209–257CrossRefPubMedGoogle Scholar
  5. 5.
    Rodan GA (1998) Mechanisms of action of bisphosphonates. Annu Rev Pharmacol Toxicol 38:375–388CrossRefPubMedGoogle Scholar
  6. 6.
    Jung A, Bisaz S, Fleisch H (1973) The binding of pyrophosphate and two diphosphonates by hydroxyapatite crystals. Calcif Tissue Res 11:269–280CrossRefPubMedGoogle Scholar
  7. 7.
    Cheong Y-K, Duncanson P, Griffiths DV, Motevalli M (2010) The impact of ligand structure on the rhenium tricarbonyl complexes from tripodal pyridine-containing chelators. Nucl Med Biol 37:677–678CrossRefGoogle Scholar
  8. 8.
    Zhou R, Deng J, Zhang M, Zhou HD, Wang YJ (2011) Association between bone mineral density and the risk of Alzheimer’s disease. J Alzheimers Dis 24:101–108PubMedGoogle Scholar
  9. 9.
    Lyell V, Henderson E, Devine M, Gregson C (2015) Assessment and management of fracture risk in patients with Parkinson’s disease. Age Ageing 44:34–41CrossRefPubMedGoogle Scholar
  10. 10.
    Torsney KM, Noyce AJ, Doherty KM, Bestwick JP, Dobson R, Lees AJ (2014) Bone health in Parkinson’s disease: a systematic review and meta-analysis. J Neurol Neurosurg Psychiatry 85:1159–1166CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    van den Bos F, Speelman AD, Samson M, Munneke M, Bloem BR, Verhaar HJJ (2012) Parkinson’s disease and osteoporosis. Age Ageing 42:156–162PubMedGoogle Scholar
  12. 12.
    Chang KH, Chung CJ, Lin CL, Sung FC, Wu TN, Kao CH (2014) Increased risk of dementia in patients with osteoporosis: a population-based retrospective cohort analysis. Age (Dordrecht) 36:967–975CrossRefGoogle Scholar
  13. 13.
    Roos PM (2014) Osteoporosis in neurodegeneration. J Trace Elem Med Biol 28:418–421CrossRefPubMedGoogle Scholar
  14. 14.
    Sato Y, Honda Y, Iwamoto J, Amano N (2013) Comparison of non-vertebral fracture between minodronate and risedronate therapy in elderly female patients with Alzheimer disease. J Musculoskelet Neuronal Interact 13:346–352PubMedGoogle Scholar
  15. 15.
    Li S, Liu B, Zhang L, Rong L (2014) Amyloid beta peptide is elevated in osteoporotic bone tissues and enhances osteoclast function. Bone 61:164–175CrossRefPubMedGoogle Scholar
  16. 16.
    Xia WF, Jung JU, Shun C, Xiong S, Xiong L, Shi XM, Mei L, Xiong WC (2013) Swedish mutant APP suppresses osteoblast differentiation and causes osteoporotic deficit, which are ameliorated by N-acetyl-l-cysteine. J Bone Miner Res 28:2122–2135CrossRefPubMedGoogle Scholar
  17. 17.
    Lane RK, Hilsabeck T, Rea SL (2015) The role of mitochondrial dysfunction in age-related diseases. Biochim Biophys Acta 1847:1387–1400CrossRefPubMedGoogle Scholar
  18. 18.
    Xu P, Xu J, Liu S, Ren G, Yang Z (2012) In vitro toxicity of nanosized copper particles in PC12 cells induced by oxidative stress. J Nanopart Res 14:1–9Google Scholar
  19. 19.
    Liu S, Han Y, Zhang T, Yang Z (2011) Protective effect of trifluoperazine on hydrogen peroxide-induced apoptosis in PC12 cells. Brain Res Bull 84:183–188CrossRefPubMedGoogle Scholar
  20. 20.
    Xu L, Xu J, Liu S, Yang Z (2014) Induction of apoptosis by antimycin A in differentiated PC12 cell line. J Appl Toxicol 34:651–657CrossRefGoogle Scholar
  21. 21.
    Han Y-g, Liu S-c, Zhang T, Yang Z (2011) Induction of apoptosis by melamine in differentiated PC12 cells. Cell Mol Neurobiol 31:65–71CrossRefPubMedGoogle Scholar
  22. 22.
    Yang X, Wang Y, Luo J, Liu S, Yang Z (2011) Protective effects of YC-1 against glutamate induced PC12 cell apoptosis. Cell Mol Neurobiol 31:303–311CrossRefPubMedGoogle Scholar
  23. 23.
    Yu L, Wang N, Zhang Y, Wang Y, Li J, Wu Q, Liu Y (2014) Neuroprotective effect of muscone on glutamate-induced apoptosis in PC12 cells via antioxidant and Ca(2+) antagonism. Neurochem Int 70:10–21CrossRefPubMedGoogle Scholar
  24. 24.
    Yan F, Wang M, Chen H, Su J, Wang X, Wang F, Xia L, Li Q (2011) Gambogenic acid mediated apoptosis through the mitochondrial oxidative stress and inactivation of Akt signaling pathway in human nasopharyngeal carcinoma CNE-1 cells. Eur J Pharmacol 652:23–32CrossRefPubMedGoogle Scholar
  25. 25.
    Lin SS, Zhu B, Guo ZK, Huang GZ, Wang Z, Chen J, Wei XJ, Li Q (2014) Bone marrow mesenchymal stem cell-derived microvesicles protect rat pheochromocytoma PC12 cells from glutamate-induced injury via a PI3K/Akt dependent pathway. Neurochem Res 39:922–931CrossRefPubMedGoogle Scholar
  26. 26.
    Liu X, Feng L, Yan M, Xu K, Yu Y, Zheng X (2010) Changes in mitochondrial dynamics during amyloid beta-induced PC12 cell apoptosis. Mol Cell Biochem 344:277–284CrossRefPubMedGoogle Scholar
  27. 27.
    Liu S-b, Zhang N, Guo Y-y, Zhao R, Shi T-y, Feng S-f, Wang S-q, Yang Q, Li X-q, Wu Y-m, Ma L, Hou Y, Xiong L-z, Zhang W, Zhao M-g (2012) G-Protein-coupled receptor 30 mediates rapid neuroprotective effects of estrogen via depression of NR2B-containing NMDA receptors. Journal of Neuroscience 32:4887–4900CrossRefPubMedGoogle Scholar
  28. 28.
    Papapoulos SE (2008) Bisphosphonates: how do they work? Best Pract Res Clin Endocrinol Metab 22:831–847CrossRefPubMedGoogle Scholar
  29. 29.
    Derenne S, Amiot M, Barille S, Collette M, Robillard N, Berthaud P, Harousseau JL, Bataille R (1999) Zoledronate is a potent inhibitor of myeloma cell growth and secretion of IL-6 and MMP-1 by the tumoral environment. J Bone Miner Res 14:2048–2056CrossRefPubMedGoogle Scholar
  30. 30.
    Paterson R (2002) Pamidronate next on list as potential cure for leishmaniasis. Lancet Infect Dis 2:515CrossRefGoogle Scholar
  31. 31.
    Kunzmann V, Bauer E, Wilhelm M (1999) Gamma/delta T-cell stimulation by pamidronate. N Engl J Med 340:737–738CrossRefPubMedGoogle Scholar
  32. 32.
    Kunzmann V, Bauer E, Feurle J, Weissinger F, Tony HP, Wilhelm M (2000) Stimulation of gamma delta T cells by aminobisphosphonates and induction of antiplasma cell activity in multiple myeloma. Blood 96:384–392PubMedGoogle Scholar
  33. 33.
    Wilhelm M, Kunzmann V, Eckstein S, Reimer P, Weissinger F, Ruediger T, Tony HP (2003) gamma delta T cells for immune therapy of patients with lymphoid malignancies. Blood 102:200–206CrossRefPubMedGoogle Scholar
  34. 34.
    Dieli F, Vermijlen D, Fulfaro F, Caccamo N, Meraviglia S, Cicero G, Roberts A, Buccheri S, D’Asaro M, Gebbia N, Salerno A, Eberl M, Hayday AC (2007) Targeting human gamma delta T cells with zoledronate and interleukin-2 for immunotherapy of hormone-refractory prostate cancer. Cancer Res 67:7450–7457CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Oldfield E (2010) Targeting isoprenoid biosynthesis for drug discovery: bench to bedside. Acc Chem Res 43:1216–1226CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Rudy CC, Hunsberger HC, Weitzner DS, Reed MN (2015) The role of the tripartite glutamatergic synapse in the pathophysiology of Alzheimer’s disease. Aging Dis 6:131–148CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Yamada K, Holth JK, Liao F, Stewart FR, Mahan TE, Jiang H, Cirrito JR, Patel TK, Hochgrafe K, Mandelkow EM, Holtzman DM (2014) Neuronal activity regulates extracellular tau in vivo. J Exp Med 211:387–393CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Li N, Liu B, Dluzen DE, Jin Y (2007) Protective effects of ginsenoside Rg2 against glutamate-induced neurotoxicity in PC12 cells. J Ethnopharmacol 111:458–463CrossRefPubMedGoogle Scholar
  39. 39.
    Ma S, Liu H, Jiao H, Wang L, Chen L, Liang J, Zhao M, Zhang X (2012) Neuroprotective effect of ginkgolide K on glutamate-induced cytotoxicity in PC 12 cells via inhibition of ROS generation and Ca(2+) influx. Neurotoxicology 33:59–69CrossRefPubMedGoogle Scholar
  40. 40.
    Sun R, Wang K, Wu D, Li X, Ou Y (2012) Original article Protective effect of paeoniflorin against glutamate-induced neurotoxicity in PC12 cells via Bcl-2/Bax signal pathway. Folia Neuropathol 3:270–276CrossRefGoogle Scholar
  41. 41.
    Emerit J, Edeas M, Bricaire F (2004) Neurodegenerative diseases and oxidative stress. Biomed Pharmacother 58:39–46CrossRefPubMedGoogle Scholar
  42. 42.
    Pi R, Li W, Lee NT, Chan HH, Pu Y, Chan LN, Sucher NJ, Chang DC, Li M, Han Y (2004) Minocycline prevents glutamate-induced apoptosis of cerebellar granule neurons by differential regulation of p38 and Akt pathways. J Neurochem 91:1219–1230CrossRefPubMedGoogle Scholar
  43. 43.
    Hausenloy D (2003) The mitochondrial permeability transition pore: its fundamental role in mediating cell death during ischaemia and reperfusion. J Mol Cell Cardiol 35:339–341CrossRefPubMedGoogle Scholar
  44. 44.
    Sousa SC, Maciel EN, Vercesi AE, Castilho RF (2003) Ca2+-induced oxidative stress in brain mitochondria treated with the respiratory chain inhibitor rotenone. FEBS Lett 543:179–183CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Wen Li
    • 1
  • Yuen-Ki Cheong
    • 2
  • Hui Wang
    • 3
  • Guogang Ren
    • 2
  • Zhuo Yang
    • 1
    Email author
  1. 1.School of Medicine, State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Tumor Microenvironment and Neurovascular RegulationNankai UniversityTianjinChina
  2. 2.Science and Technology Research InstituteUniversity of HertfordshireHatfield, HertsUK
  3. 3.College of Life SciencesNankai UniversityTianjinChina

Personalised recommendations