, Volume 19, Issue 5, pp 789–800 | Cite as

17β-Estradiol protects against apoptosis induced by levofloxacin in rat nucleus pulposus cells by upregulating integrin α2β1

  • Si-Dong Yang
  • Lei Ma
  • Ti-Xin Gu
  • Wen-Yuan Ding
  • Feng Zhang
  • Yong Shen
  • Ying-Ze Zhang
  • Da-Long Yang
  • Di Zhang
  • Ya-Peng Sun
  • Yan-Li Song
Original Paper


Levofloxacin has been reported to have cytotoxicity to chondrocytes in vitro. And 17β-estradiol has been widely studied for its protective effects against cell apoptosis. Based on apoptotic cell model induced by levofloxacin, the purpose of this study was to explore the mechanism by which 17β-estradiol protects rat nucleus pulposus cells from apoptosis. Inverted phase-contrast microscopy, flow cytometry, and caspase-3 activity assay were used to find that levofloxacin induced marked apoptosis, which was abolished by 17β-estradiol. Interestingly, estrogen receptor antagonist, ICI182780, and functional blocking antibody to α2β1 integrin, both prohibited the effect of 17β-estradiol. Simultaneously, levofloxacin decreased cellular binding ability to type II collagen, which was also reversed by 17β-estradiol. Furthermore, western blot and real-time quantitative PCR were used to find that integrin α2β1 was responsible for estrogen-dependent anti-apoptosis, which was time–response and dose–response effect. 17β-estradiol was proved for the first time to protect rat nucleus pulposus cells against levofloxacin-induced apoptosis by upregulating integrin α2β1 signal pathway.


17β-Estradiol Levofloxacin Nucleus pulposus Integrin α2β1 Apoptosis 





  1. 1.
    Frymoyer JW, Cats-Baril WL (1991) An overview of the incidence and costs of low back pain. Orthop Clin N Am 22:263–271Google Scholar
  2. 2.
    Sakai Daisuke (2008) Future perspectives of cell-based therapy for intervertebral disc disease. Eur Spine J 17(Suppl 4):S452–S458. doi: 10.1007/s00586-008-0743-5 CrossRefGoogle Scholar
  3. 3.
    Antoniou J, Steffen T, Nelson F, Winterbottom N, Hollander AP, Poole RA, Aebi M, Alini M (1996) The human lumbar intervertebral disc: evidence for changes in the biosynthesis and denaturation of the extracellular matrix with growth, maturation, ageing, and degeneration. J Clin Invest 98:996–1003PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Alini M, Roughley PJ, Antoniou J, Stoll T, Aebi M (2002) A biological approach to treating disc degeneration: not for today, but may be for tomorrow. Eur Spine J 11:S215–S220PubMedCentralPubMedGoogle Scholar
  5. 5.
    Buckwalter JA (1995) Aging and degeneration of the human intervertebral disc. Spine 20:1307–1314PubMedGoogle Scholar
  6. 6.
    Zhou Guang-Qian, Yang Fan, Victor VL (2008) Molecular and cellular biology of the intervertebral disc and the use of animal models. Curr Ortho 22:267–273CrossRefGoogle Scholar
  7. 7.
    Le Maitre CL, Freemont AJ, Hoyland JA (2007) Accelerated cellular senescence in degenerate intervertebral discs: a possible role in the pathogenesis of intervertebral disc degeneration. Arthritis Res Ther 9:R45PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Boos N, Weissbach S, Rohrbach H, Weiler C, Spratt KF, Nerlich AG (2002) Classification of age-related changes in lumbar intervertebral discs: 2002 volvo award in basic science. Spine 27:2631–2644PubMedCrossRefGoogle Scholar
  9. 9.
    Bozzo C, Graziola F, Chiocchetti A, Canonico PL (2010) Estrogen and β-amyloid toxicity: role of integrin and PI3-K. Mol Cell Neurosci 45:85–91PubMedCrossRefGoogle Scholar
  10. 10.
    Nelson K, Helmstaedter V, Moreau C, Lage H (2008) Estradiol, tamoxifen and ICI182,780 alter α3 and β1 integrin expression and laminin-1 adhesion in oral squamous cell carcinoma cell cultures. Oral Oncol 44:94–99PubMedCrossRefGoogle Scholar
  11. 11.
    Zandi PP, Carlson MC, Plassman BL, Welsh-Bohmer KA, Mayer LS, Steffens DC, Breitner JC, Cache County Memory Study Investigators (2002) Cache County Memory Study Investigators: hormone replacement therapy and incidence of Alzheimer disease in older women: the Cache County Study. JAMA 288:2123–2129PubMedCrossRefGoogle Scholar
  12. 12.
    Craig MC, Maki PM, Murphy DG (2005) The Women’s health initiative memory study: findings and implications for treatment. Lancet Neurol 4:190–194PubMedCrossRefGoogle Scholar
  13. 13.
    Honda K, Sawada H, Kihara T, Urushitani M, Nakamizo T, Akaike A, Shimohama S (2000) Phosphatidylinositol 3-kinase mediates neuroprotection by estrogen in cultured cortical neurons. J Neurosci Res 60:321–327PubMedCrossRefGoogle Scholar
  14. 14.
    Zhang L, Rubinow DR, Xaing G, Li BS, Chang YH, Maric D, Barker JL, Ma W (2001) Estrogen protects against beta-amyloid-induced neurotoxicity in rat hippocampal neurons by activation of Akt. NeuroReport 12:1919–1923PubMedCrossRefGoogle Scholar
  15. 15.
    Hynes RO (1987) Integrins: a family of cell surface receptors. Cell 48:549–554PubMedCrossRefGoogle Scholar
  16. 16.
    Boudreau N, Sympson CJ, Werb Z, Bissell MJ (1995) Suppression of ICE and apoptosis in mammary epithelial cells by extracellular matrix. Science 267:891–893PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Lee JW, Juliano R (2004) Mitogenic signal transduction by integrin- and growth factor receptor-mediated pathways. Mol Cells 17:188–202PubMedGoogle Scholar
  18. 18.
    Harburger DS, Calderwood DA (2009) Integrin signalling at a glance. J Cell Sci 122:159–163PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Streuli CH (2009) Integrins and cell-fate determination. J Cell Sci 122:171–177PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Humphries JD, Byron A, Humphries MJ (2006) Integrin ligands at a glance. J Cell Sci 119:3901–3903PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Huhtala M, Heino J, Casciari D, De Luise A, Johnson MS (2005) Integrin evolution: insights from ascidian and teleost fish genomes. Matrix Biol 24:83–95PubMedCrossRefGoogle Scholar
  22. 22.
    Johnson MS, Lu N, Denessiouk K, Heino J, Gullberg D (2009) Integrins during evolution: evolutionary trees and model organisms. Biochim Biophys Acta 1788:779–789PubMedCrossRefGoogle Scholar
  23. 23.
    Leitinger B, Hohenester E (2007) Mammalian collagen receptors. Matrix Biol 26:146–155PubMedCrossRefGoogle Scholar
  24. 24.
    Takada Y, Ye X, Simon S (2007) The integrins. Genome Biol 8:215PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Li J, Tan H, Dong X, Xu Z, Shi C, Han X, Jiang H, Krissansen GW, Sun X (2007) Antisense integrin αV and β3 gene therapy suppresses subcutaneously implanted hepatocellular carcinomas. Dig Liver Dis 39:557–565PubMedCrossRefGoogle Scholar
  26. 26.
    Sakamoto Y, Ogita H, Hirota T, Kawakatsu T, Fukuyama T, Yasumi M, Kanzaki N, Ozaki M, Takai Y (2006) Interaction of integrin avb3 with nectin. Implication in cross-talk between cell–matrix and cell–cell junctions. J Biol Chem 281:19631–19644PubMedCrossRefGoogle Scholar
  27. 27.
    Sakamoto Y, Ogita H, Komura H, Takai Y (2008) Involvement of nectin in inactivation of integrin αvβ3 after the establishment of cell–cell adhesion. J Biol Chem 283:496–505PubMedCrossRefGoogle Scholar
  28. 28.
    Sun H, Hu K, Wu M, Xiong J, Yuan L, Tang Y, Yang Y, Liu H (2011) Contact by melanoma cells causes malignant transformation of human epithelial-like stem cells via alpha V integrin activation of transforming growth factor β1 signaling. Exp Biol Med (Maywood) 236:352–365CrossRefGoogle Scholar
  29. 29.
    Canonici A, Steelant W, Rigot V, Khomitch-Baud A, Boutaghou-Cherid H, Bruyneel E, Van Roy F, Garrouste F, Pommier G, André F (2008) Insulin-like growth factor-I receptor, E-cadherin and αv integrin forma dynamic complex under the control of alpha-catenin. Int J Cancer 122:572–582PubMedCrossRefGoogle Scholar
  30. 30.
    Vellon L, Menendez JA, Lupu R (2006) A bidirectional αvβ3 integrin-ERK1/ERK2 MAPK connection regulates the proliferation of breast cancer cells. Mol Carcinog 5:795–804CrossRefGoogle Scholar
  31. 31.
    Ou J, Luan W, Deng J, Sa R, Liang H (2012) αV Integrin induces multicellular radioresistance in human nasopharyngeal carcinoma via activating SAPK/JNK pathway. PLoS ONE 7(6):e38737. doi: 10.1371/journal.pone.0038737 PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    Byzova Tatiana V, Kim Wes, Midura Ronald J, Plow Edward F (2000) Activation of integrin αVβ3 regulates cell adhesion and migration to bone sialoprotein. Exp Cell Res 254:299–308PubMedCrossRefGoogle Scholar
  33. 33.
    Sendzik J, Shakibaei M, Scha¨fer-Korting M, Stahlmann R (2005) Fluoroquinolones cause changes in extracellular matrix, signalling proteins, metalloproteinases and caspase-3 in cultured human tendon cells. Toxicology 212:24–36PubMedCrossRefGoogle Scholar
  34. 34.
    Costa-Rodrigues J, Martins EG, Fernandes MH (2012) Induced osteoclastogenesis by fluoroquinolones in unstimulated and stimulated human osteoclast precursor cells. Bone 51:17–27PubMedCrossRefGoogle Scholar
  35. 35.
    Zhao Chang-Qing, Liu Da, Li Hai, Jiang Lei-Sheng, Dai Li-Yang (2007) Interleukin-1β enhances the effect of serum deprivation on rat annular cell apoptosis. Apoptosis 12:2155–2161PubMedCrossRefGoogle Scholar
  36. 36.
    Sobrino A, Mata M, Laguna-Fernandez A, Novella S, Oviedo PJ, García-Pérez MA, Tarín JJ, Cano A, Hermenegildo C (2009) Estradiol stimulates vasodilatory and metabolic pathways in cultured human endothelial cells. PLoS ONE 4(12):e8242. doi: 10.1371/journal.pone.0008242 PubMedCentralPubMedCrossRefGoogle Scholar
  37. 37.
    Zhang Z, Vuori K, Reed JC, Ruoslahti E (1995) The alpha5beta1 integrin supports survival of cells on fibronectin and up-regulates Bcl-2 expression. Proc Natl Acad Sci USA 92:6161–6165PubMedCentralPubMedCrossRefGoogle Scholar
  38. 38.
    De Toni-Costes F, Despeaux M, Bertrand J, Bourogaa E, Ysebaert L, Payrastre B, Racaud-Sultan C (2010) A New alpha5beta1 integrin-dependent survival pathway through GSK3beta activation in leukemic cells. PLoS ONE 5(3):e9807. doi: 10.1371/journal.pone.0009807 PubMedCentralPubMedCrossRefGoogle Scholar
  39. 39.
    Gibson RM, Craig SE, Heenan L, Tournier C, Humphries MJ (2005) Activation of integrin α5β1 delays apoptosis of Ntera2 neuronal cells. Mol Cell Neurosci 28:588–598PubMedCrossRefGoogle Scholar
  40. 40.
    Loeser RF, Sadiev S, Tan L, Goldring MB (2000) Integrin expression by primary and immortalized human chondrocytes: evidence of a differential role for alpha1beta1 and alpha2beta1 integrins in mediating chondrocyte adhesion to types II and VI collagen. Osteoarthr Cartil 8:96–105PubMedCrossRefGoogle Scholar
  41. 41.
    Paoli P, Giannoni E, Chiarugi P (2013) Anoikis molecular pathways and its role in cancer progression. Biochim Biophys Acta 1833:3481–3498PubMedCrossRefGoogle Scholar
  42. 42.
    Zhong X, Rescorla FJ (2012) Cell surface adhesion molecules and adhesion-initiated signaling: understanding of anoikis resistance mechanisms and therapeutic opportunities. Cell Signal 24:393–401PubMedCrossRefGoogle Scholar
  43. 43.
    Meredith JE Jr, Fazeli B, Schwartz MA (1993) The extracellular matrix as a cell survival factor. Mol Biol Cell 4:953–961PubMedCentralPubMedCrossRefGoogle Scholar
  44. 44.
    Frisch SM, Francis H (1994) Disruption of epithelial cell-matrix interactions induces apoptosis. J Cell Biol 124:619–626PubMedCrossRefGoogle Scholar
  45. 45.
    Chiarugi P, Giannoni E (2008) Anoikis: a necessary death program for anchorage-dependent cells. Biochem Pharmacol 76:1352–1364PubMedCrossRefGoogle Scholar
  46. 46.
    Zoppi N, Barlati S, Colombi M (2008) FAK-independent alphavbeta3 integrin-EGFR complexes rescue from anoikis matrix-defective fibroblasts. Biochim Biophys Acta 1783(6):1177–1188PubMedCrossRefGoogle Scholar
  47. 47.
    Ludovic Carduner, Cédric R. Picot, Johanne Leroy-Dudal, Lyvia Blay, Sabrina Kellouche, Franck Carreiras (2013) Cell cycle arrest or survival signaling through αv integrins, activation of PKC and ERK1/2 lead to anoikis resistance of ovarian cancer spheroids. Exp Cell Res (in press)
  48. 48.
    Rohwer N, Welzel M, Daskalow K, Pfander D, Wiedenmann B, Detjen K, Cramer T (2008) Hypoxia-inducible factor 1α mediates anoikis resistance via suppression of α5 integrin. Cancer Res 68(24):10113–10120PubMedCrossRefGoogle Scholar
  49. 49.
    Chiarugi P, Giannoni E (2008) Anoikis: a necessary death program for anchorage-dependent cells. Biochem Pharmacol 76:1352–1364PubMedCrossRefGoogle Scholar
  50. 50.
    Frisch SM, Screaton RA (2001) Anoikis mechanisms. Curr Opin Cell Biol 13:555–562PubMedCrossRefGoogle Scholar
  51. 51.
    Gilmore AP (2005) Anoikis. Cell Death Differ 12(Suppl. 2):1473–1477PubMedCrossRefGoogle Scholar
  52. 52.
    Taddei ML, Giannoni E, Fiaschi T, Chiarugi P (2012) Anoikis: an emerging hallmark in health and diseases. J Pathol 226:380–393PubMedCrossRefGoogle Scholar
  53. 53.
    Deng Y, Chen B, Qi Y, Magdalou J, Wang H, Chen L (2011) The effects of levofloxacin on rabbit anterior cruciate ligament cells in vitro. Toxicol Appl Pharmacol 257(1):67–73PubMedCrossRefGoogle Scholar
  54. 54.
    Tan Y, Lu K, Deng Y, Cao H, Chen B, Wang H, Magdalou J, Chen L (2012) The effects of levofloxacin on rabbit fibroblast-like synoviocytes in vitro. Toxicol Appl Pharmacol 265:175–180PubMedCrossRefGoogle Scholar
  55. 55.
    Sheng Zhi-Guo, Huang Wei, Liu Yu-Xiang, Yuan Ye, Zhu Ben-Zhan (2013) Ofloxacin induces apoptosis via β1 integrin-EGFR-Rac1-Nox2 pathway in microencapsulated chondrocytes. Toxicol Appl Pharmacol 267:74–87PubMedCrossRefGoogle Scholar
  56. 56.
    Sawhney RS, Sharma B, Humphrey LE, Brattain MG (2003) Integrin alpha2 and extracellular signal-regulated kinase are functionally linked in highly malignant autocrine transforming growth factor-alpha-driven colon cancer cells. J Biol Chem 278:19861–19869PubMedCrossRefGoogle Scholar
  57. 57.
    Liang CC, Chen HC (2001) Sustained activation of extracellular signal-regulated kinase stimulated by hepatocyte growth factor leads to integrin alpha 2 expression that is involved in cell scattering. J Biol Chem 276:21146–21152PubMedCrossRefGoogle Scholar
  58. 58.
    Zutter MM, Painter AD, Yang X (1999) The Megakaryocyte/Platelet-specificenhancer of the alpha2beta1 integrin gene: two tandem AP1 sites and the mitogen-activated protein kinase signaling cascade. Blood 93:1600–1611PubMedGoogle Scholar
  59. 59.
    Risbud Makarand V, Guttapalli Asha, Albert Todd J, Shapiro Irving M (2005) Hypoxia activates MAPK activity in rat nucleus pulposus cells: regulation of integrin expression and cell survival. SPINE 30:2503–2509PubMedCrossRefGoogle Scholar
  60. 60.
    Hennessy AB, Harvey BJ, Healy V (2005) 17-Estradiol rapidly stimulates c-fos expression via the MAPK pathway in T84 cells. Mol Cell Endocrinol 229:39–47PubMedCrossRefGoogle Scholar
  61. 61.
    Wesierska-Gadek J, Schreiner T, Gueorguieva M, Ranftler C (2006) Phenol red reduces ROSC mediated cell cycle arrest and apoptosis in human MCF-7 cells. J Cell Biochem 98:1367–1379PubMedCrossRefGoogle Scholar
  62. 62.
    Levin ER (2001) Cell localization, physiology, and nongenomic actions of estrogen receptors. J Appl Physiol 91:1860–1867PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Si-Dong Yang
    • 1
    • 4
  • Lei Ma
    • 1
    • 4
  • Ti-Xin Gu
    • 2
  • Wen-Yuan Ding
    • 1
    • 4
  • Feng Zhang
    • 3
  • Yong Shen
    • 1
    • 4
  • Ying-Ze Zhang
    • 1
    • 4
  • Da-Long Yang
    • 1
    • 4
  • Di Zhang
    • 1
    • 4
  • Ya-Peng Sun
    • 1
    • 4
  • Yan-Li Song
    • 1
  1. 1.Department of Spine SurgeryThe Third Hospital of Hebei Medical UniversityShijiazhuangChina
  2. 2.Department of VIP WardThe First Hospital of Hebei Medical UniversityShijiazhuangChina
  3. 3.Department of Rehabilitation MedicineThe Third Hospital of Hebei Medical UniversityShijiazhuangChina
  4. 4.Hebei Provincial Key Laboratory of Orthopedic BiomechanicsShijiazhuangChina

Personalised recommendations