Inhibition of matrix metalloproteinase-14 in osteosarcoma cells by clodronate.
BACKGROUND
Bisphosphonates reduce the bone metastasis formation and angiogenesis but the exact molecular mechanisms involved are unclear. Progelatinase A (proMMP-2; 78 KDa) is activated up during the tumor spread and metastasis by a cell surface-associated matrix metalloproteinase (membrane-type matrix metalloproteinase [MT1-MMP] or MMP-14).


METHODS
We evaluated the effects of a bisphosphonate (clodronate) on MT1-MMP mRNA expression and protein production, catalytic activity and proteolytic activation of proMMP-2 by cultured human MG-63 osteosarcoma cells.


RESULTS
Clodronate, at therapeutically attainable noncytotoxic concentrations, dose-dependently inhibited phorbol myristic acetate (PMA)-induced proteolytic activation of proMMP-2 by human MG-63 osteosarcoma cells. Clodronate also downregulated the PMA-induced expression of MT1-MMP mRNA and protein production in human MG-63 osteosarcoma cells, as evidenced by Northern analysis and fluorescent immunohistochemistry. Furthermore, clodronate inhibited directly and dose-dependently MT1-MMP activity, and the MT1-MMP inhibition by clodronate was reduced in the presence of an increased (5 mM) Ca(2+) concentrations when compared to physiological (1 mM) Ca(2+) concentrations.


CONCLUSIONS
We conclude that (1) the extracellular/cell-associated mechanism of bisphosphonate involves inhibition of MT1-MMP catalytic activity eventually by chelation, and that (2) intracellular mechanism involves downregulation of induced MT1-MMP mRNA and protein expression. The inhibition and downregulation of MT1-MMP by clodronate can be related to their ability to reduce MG-63 osteosarcoma cell invasion and spread. These findings may, at least in part, explain at molecular level the antitumor and antibone resorption activities of clodronate observed in clinical studies.
Heikkilä
Pia
P
Department of Oral and Maxillofacial Diseases, Helsinki University Central Hospital, Institute of Dentistry, University of Helsinki, Helsinki, Finland.
Teronen
Olli
O
Hirn
Martti Y
MY
Sorsa
Timo
T
Tervahartiala
Taina
T
Salo
Tuula
T
Konttinen
Yrjö T
YT
Halttunen
Tuula
T
Moilanen
Merja
M
Hanemaaijer
Roeland
R
Laitinen
Minna
M
D001665:Binding Sites / Q000187:drug effects; D015152:Blotting, Northern; D004002:Clodronic Acid / Q000494:pharmacology*; D017364:Collagenases / Q000096:biosynthesis; D017077:Culture Media, Conditioned; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004792:Enzyme Precursors / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D018093:Gelatinases / Q000378:metabolism; D006801:Humans; D053509:Matrix Metalloproteinase 13; D020778:Matrix Metalloproteinase 2 / Q000096:biosynthesis / Q000235:genetics; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D012516:Osteosarcoma / Q000201:enzymology*; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D017077:Culture Media, Conditioned; D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D012333:RNA, Messenger; D011994:Recombinant Proteins; D004002:Clodronic Acid; D017364:Collagenases; D018093:Gelatinases; C507079:MMP13 protein, human; D053509:Matrix Metalloproteinase 13; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; C069860:progelatinase; D020778:Matrix Metalloproteinase 2; D013755:Tetradecanoylphorbol Acetate