Clinical & Experimental Metastasis

, Volume 25, Issue 4, pp 437–444 | Cite as

Human breast adenocarcinoma (MDA-231) and human lung squamous cell carcinoma (Hara) do not have the ability to cause bone resorption by themselves during the establishment of bone metastasis

  • Amy Tomita
  • Tatsuhiko Kasaoka
  • Takashi Inui
  • Minako Toyoshima
  • Hiroko Nishiyama
  • Hiromasa Saiki
  • Haruo Iguchi
  • Motowo Nakajima
Research Paper


Osteolysis is an important process in the establishment of bone metastasis. The role which cancer cells play in this process is not fully understood. In this study, we first established a reproducible in vivo bone metastasis model using two types of tumor cells, human breast adenocarcinoma (MDA-231) and human lung squamous cell carcinoma (Hara cells), and examined in vitro characteristics of the tumor cells. Tumor cells injected into the left heart ventricle of nude mice preferentially metastasized to bone, 6 weeks after the inoculation. Histological observation of the bone metastatic lesion showed that tumor cells invaded the bone marrow, and osteoclasts adjacent to fibroblasts were actively resorbing the bone matrix. In vitro analysis of the tumor cells showed that MDA-231 cells express cathepsin K, matrix metalloproteinase 9 (MMP-9), and membrane type-1 matrix metalloproteinase (MT1-MMP), all of which are believed to play an important role in osteoclastic bone resorption. In contrast, Hara cells do not express cathepsin K and MT1-MMP. MMP-9 was expressed at a low level. To assess the osteolytic activity of the tumor cells, an in vitro pit assay was performed. The rabbit osteoclasts formed numerous pits on a dentin slice after 18 h of incubation, whereas tumor cells by themselves did not. Taken together, we conclude that MDA-231 and Hara cells, which metastasize to the bone in vivo, do not have enough ability to achieve bone resorption by themselves, but rather achieve it through activation of fibroblast like cells and osteoclasts.


Bone metastasis Breast cancer Cathepsin K Lung cancer MMP-9 MT1-MMP 



Dulbecco’s modified Eagle’s medium


Minimum essential medium


Matrix metalloproteinase 9


Membrane type-1 matrix metalloproteinase


Phenylmethanesulfonyl fluoride


Parathyroid hormone related protein


Reverse transcriptase–polymerase chain reaction


Sodium dodecyl sulfate-polyacrylamide gel electrophoresis


  1. 1.
    Tezuka K, Tezuka Y, Maejima A et al (1994) Molecular cloning of a possible cysteine proteinase predominantly expressed in osteoclasts. J Biol Chem 269:1106–1109PubMedGoogle Scholar
  2. 2.
    Inaoka T, Bilbe G, Ishibashi O et al (1995) Molecular cloning of human cDNA for Cathepsin K: novel cysteine proteinase predominantly expressed in bone. Biochem Biophys Res Commun 206:89–96PubMedCrossRefGoogle Scholar
  3. 3.
    Drake F, Dodds RA, James IE et al (1996) Cathepsin K, but not Cathepsin B, L, or S, is abundantly expressed in human osteoclasts. J Biol Chem 271:12511–12516PubMedCrossRefGoogle Scholar
  4. 4.
    Bromme D, Okamoto K, Wang BB et al (1996) Human cathepsin O2, a matrix protein-degrading cysteine protease expressed in osteoclasts. J Biol Chem 271:2126–2132PubMedCrossRefGoogle Scholar
  5. 5.
    Inui T, Ishibashi O, Inaoka T et al (1997) Cathepsin K antisense oligodeoxynucleotide inhibits osteoclastic bone resorption. J Biol Chem 272:8109–8112PubMedCrossRefGoogle Scholar
  6. 6.
    Saftig P, Hunziker E, Wehmeyer O et al (1998) Impaired osteoclastic bone resorption leads to osteopetrosis in cathepsin K-deficient mice. Proc Natl Acad Sci USA 95:13453–13458PubMedCrossRefGoogle Scholar
  7. 7.
    Tezuka K, Nemoto K, Tesuka Y et al (1994) Identification of matrix metalloproteinase 9 in rabbit osteoclasts. J Biol Chem 269:15006–15009PubMedGoogle Scholar
  8. 8.
    Okada Y, Naka K, Kawamura K et al (1995) Localization of matrix metalloproteinase 9 (92-kilodalton gelatinase/type IV collagenase = gelatinase B) in osteoclasts: implication for bone resorption. Lab Invest 72:311–322PubMedGoogle Scholar
  9. 9.
    Garnero P, Borel O, Byrjalsen I et al (1998) The collagenolyitic activity of cathepsin K is unique amongst mammalian proteinases. J Biol Chem 273:32347–32352PubMedCrossRefGoogle Scholar
  10. 10.
    Sato H, Takino T, Okada Y et al (1994) A matrix metalloproteinase expressed on the surface of invasive cells. Nature 370:61–65PubMedCrossRefGoogle Scholar
  11. 11.
    Cao J, Sato H, Takino T et al (1995) The c-terminal region of membrane type matrix metalloproteinase is a functional transmembrane domain required for pro-gelatinase A activation. J Biol Chem 270:801–805PubMedCrossRefGoogle Scholar
  12. 12.
    Inada M, Miyaura C (2001) Bone metabolism and MMPs. Bone 15:123–129Google Scholar
  13. 13.
    Sato T, del Carmen Ovejero M, Hou P et al (1997) Identification of the membrane-type matrix proteinase MT1-MMP in osteoclasts. J Cell Sci 110:589–598PubMedGoogle Scholar
  14. 14.
    Irie k, Tsuruga E, Sakakura Y et al (2001) Immunohistochemical localization of membrane type 1-matrix metalloproteinase (MT1-MMP) in osteoclasts in vivo. Tissue cell 33:478–482PubMedCrossRefGoogle Scholar
  15. 15.
    Sasaki A, Boyce BF, Story B et al (1995) Bisphosphonate risedronate reduces metastatic human breast cancer burden in bone in nude mice. Cancer Res 55:3551–3557PubMedGoogle Scholar
  16. 16.
    Littlewood-Evans A, Kokubo T, Ishibashi O et al (1997) Localization of Cathepsin K in human osteoclasts by in situ hybridization and immunohistochemistry. Bone 20:81–86PubMedCrossRefGoogle Scholar
  17. 17.
    Nakajima M, Welch DR, Wynn DM et al (1993) Serum and plasma Mr 92,000 progelatinase levels correlate with spontaneous metastasis of rat 13762NF mammary adenocarcinoma. Cancer Res 53:5802–5807PubMedGoogle Scholar
  18. 18.
    Tezuka K, Sato T, Kamioka et al (1992) Identification of osteopontin in isolated rabbit osteoclasts. Biochem Biophys Res Commun 186:911–917PubMedCrossRefGoogle Scholar
  19. 19.
    Takada Y, Kusuda K, Hiura K et al (1992) A simple method to assess osteoclast-mediated bone resorption using unfractionated bone cells. Bone Miner 17:347–359PubMedCrossRefGoogle Scholar
  20. 20.
    Iguchi H, Tanaka S, Ozawa Y et al (1996) An experimental model of bone metastasis by human lung cancer cells: the role of parathyroid hormone-related protein in bone metastasis. Cancer Res 56:4040–4043PubMedGoogle Scholar
  21. 21.
    Ishibashi O, Niwa S, Kadoyama K et al (2006) MMP-9 antisense oligodeoxynucleotide exerts an inhibitory effect on osteoclastic bone resorption by suppressing cell migration. Life Sci 79:1657–1660PubMedCrossRefGoogle Scholar
  22. 22.
    Inui T, Ishibashi O, Origane Y et al (1999) Matrix metalloproteinase and lysosomal cysteine proteases in osteoclasts contribute to bone resorption through distinct modes of action. Biochem Biophys Res Commun 258:173–178PubMedCrossRefGoogle Scholar
  23. 23.
    Sato T, Forged NT, Delaisse JM (1998) The migration of purified osteoclasts through collagen is inhibited by matrix metalloproteinase inhibitors. J Bone Miner Res 13:59–66PubMedCrossRefGoogle Scholar
  24. 24.
    Engsig MT, Chen QJ, Vu TH et al (2000) Matrix metalloproteinase and vascular endothelial growth factor are essential for osteoclast recruitment into developing long bones. J Cell Biol 151:879–889PubMedCrossRefGoogle Scholar
  25. 25.
    Boyde A, Maconnachie E, Reid SA et al (1986) Scanning electron microscopy in bone pathology: review of methods, potential and applications. Scan Electron Microsc 4:1537–155Google Scholar
  26. 26.
    Suda T, Takahashi N, Udagawa N et al (1999) Modulation of osteoclast differentiation and function by the new member of the tumor necrosis factor receptor and ligand families. Endocr Rev 20:345–357PubMedCrossRefGoogle Scholar
  27. 27.
    Thomas RJ, Guise TA, Yin JJ et al (1999) Breast cancer cells interact with osteoclasts to support osteoclastic formation. Endocrinology 140:4451–4458PubMedCrossRefGoogle Scholar
  28. 28.
    Guise TA, Yin JJ, Taylor SD et al (1996) Evidence for a casual role of parathyroid hormone-related protein in the pathogenesis of human breast cancer-mediated osteolysis. J Clin Invest 98:1544–1549PubMedCrossRefGoogle Scholar
  29. 29.
    Bendre MS, Gaddy-Kurten D, Mon-Foote T et al (2002) Expression of Interleukin 8 and not parathyroid hormone-related protein by human breast cancer cells correlates with bone metastasis in vivo. Cancer Res 62:5571–5579PubMedGoogle Scholar
  30. 30.
    Iguchi H, Onuma E, Sato K et al (2001) Involvement of parathyroid hormone-related protein in experimental cachexia induced by a human lung cancer-derived cell line established from a bone metastasis. Int J Cancer 94:24–27PubMedCrossRefGoogle Scholar
  31. 31.
    Bendre MS, Margulies AG, Walser B et al (2005) Tumor-derived interleukin-8 simulates osteolysis independent of the receptor activator of nuclear factor-κB ligand pathway. Cancer Res 65:11001–11009PubMedCrossRefGoogle Scholar
  32. 32.
    Bendre MS, Montague DC, Peery T et al (2003) Interleukin-8 stimulation of osteoclastogenesis and bone resorption is a mechanism for the increased osteolysis of metastatic bone disease. Bone 33:28–37PubMedCrossRefGoogle Scholar
  33. 33.
    Perderson L, Winding B, Foged NT et al (1999) Identification of breast cancer cell line-derived paracrine factors that stimulate osteoclast activity. Cancer Res 59:5849–5855Google Scholar
  34. 34.
    Mancino AT, Klimberg VS, Yamamoto M et al (2001) Breast cancer increases osteoclastogenesis by secreting M-CSF and upregulating RANKL in stromal cells. J Surg Res 100:18–24PubMedCrossRefGoogle Scholar
  35. 35.
    Gallet M, Sevenet N, Dupont C et al (2004) Breast cancer cell line MDA-MB-231 exerts a potent and direct ant-apoptotic effect on mature osteoclasts. Biochem Biophys Res Commun 319:690–696PubMedCrossRefGoogle Scholar
  36. 36.
    Ohshiba T, Miyaura C, Inada M et al (2003) Role of RANKL-induced osteoclast formation and MMP-dependent matrix degradation by breast cancer metastasis. Br J Cancer 88:1318–1326PubMedCrossRefGoogle Scholar
  37. 37.
    Sahara T, Itho K, Debari K et al (2003) Specific biological functions of vacuolar-type H+-ATPase and lysomal cysteine proteinase, cathepsin K, in osteoclasts. Anal Rec 270A:152–161CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Amy Tomita
    • 1
    • 2
  • Tatsuhiko Kasaoka
    • 1
  • Takashi Inui
    • 1
    • 3
  • Minako Toyoshima
    • 1
  • Hiroko Nishiyama
    • 1
  • Hiromasa Saiki
    • 1
  • Haruo Iguchi
    • 4
    • 5
  • Motowo Nakajima
    • 1
    • 6
  1. 1.Discovery BiologyNovartis Institute for BioMedical ResearchTsukubaJapan
  2. 2.Showa Women’s UniversityTokyoJapan
  3. 3.Osaka Prefecture UniversityOsakaJapan
  4. 4.Division of Tumor Dynamics, Institute for Clinical ResearchNational Kyushu Cancer CenterMinami-kuJapan
  5. 5.Shikoku Cancer CenterEhimeJapan
  6. 6.Johnson & JohnsonTokyoJapan

Personalised recommendations