Abstract
Doxycycline, a tetracycline derivative, has many properties in addition to its antibiotic activity, including inhibition of matrix metalloproteinases (MMPs) and the ability to chelate divalent cations including Ca2+. It has been shown to inhibit endothelial cell growth in vitro, and reduce the development of experimental tumours, especially bone metastasis in a model of breast cancer. We examined the effects of doxycycline on angiogenesis in the chicken chorioallantoic membrane (CAM) model, and showed that doxycycline will cause loss of the chorionic plexus in CAMs when applied at day 8 of incubation, and the duration of this inhibition was dose-dependent. Repeated doses prolonged the inhibition, but following removal of the doxycycline there was rapid recovery of the chorionic plexus. The effects of doxycycline are in part mimicked by the MMP inhibitor 1,10-phenanthroline, and more closely by the Ca2+-chelating agent EGTA. Doxycycline was equally effective in causing loss of the chorionic plexus by day 11 in CAMs, a time at which the blood vessels are established. Doxycycline has important potential as an antiangiogenic treatment. It is capable of inhibiting angiogenesis in an in vivo model, including the removal of comparatively mature endothelial cells. The response is sensitive to the dosing regimen and the effect is rapidly reversible.
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Amin AR, Attur MG, Thakker GD, Patel PD, Vyas PR, Patel RN, Patel IR, Abramson SB (1996) A novel mechanism of action of tetracyclines: effects on nitric oxide synthases. Proc Natl Acad Sci U S A 93:14014
Anand-Apte B, Pepper MS, Voest E, Montesano R, Olsen B, Murphy G, Apte SS, Zetter B (1997) Inhibition of angiogenesis by tissue inhibitor of metalloproteinase-3. Invest Ophthalmol Vis Sci 38:817
Duivenvoorden WC, Hirte HW, Singh G (1997) Use of tetracycline as an inhibitor of matrix metalloproteinase activity secreted by human bone-metastasizing cancer cells. Invasion Metastasis 17:312
Duivenvoorden WCM, Popovir S, Lhoták S, Seidlitz E, Hirte HW, Tozer RG, Singh G (2002) Doxycycline decreases tumor burden in a bone metastasis model of human breast cancer. Cancer Res 62:1588
Eliceiri BP (2001) Integrin and growth factor receptor crosstalk. Circ Res 89:1104
Fife RS, Sledge GW Jr (1995) Effects of doxycycline on in vitro growth, migration, and gelatinase activity of breast cancer cells. J Lab Clin Med 125:407
Fife RS, Sledge GW Jr, Roth BJ, Proctor C (1998) Effects of doxycycline on human prostate cancer cells in vitro. Cancer Lett 127:37
Fife RS, Sledge GW Jr, Sissons S, Zerler B (2000) Effects of tetracyclines on angiogenesis in vitro. Cancer Lett 153:75
Gatto C, Rieppi M, Borsotti P, Innocenti S, Ceruti R, Drudis T, Scanziani E, Casazza AM, Taraboletti G, Giavazzi R (1999) BAY 12-9566, a novel inhibitor of matrix metalloproteinases with antiangiogenic activity. Clin Cancer Res 5:3606
Gilbertson-Beadling S, Powers EA, Stamp-Cole M, Scott PS, Wallace J, Copland G, Petzold M, Mitchell M, Ledbetter S, Poorman R, Wilks JW, Fisher C (1995) The tetracycline analogues minocycline and doxycycline inhibit angiogenesis in vitro by a non-metalloproteinase-dependent manner. Cancer Chemother Pharmacol 36:418
Ginns LC, Roberts DH, Mark EJ, Brusch JL, Marler JJ (2003) Pulmonary capillary hemangiomatosis with atypical endotheliomatosis. Successful antiangiogenic therapy with doxycycline. Chest 124:2017
Gelette J, Sandberg S, Hopen G, Solberg CO (1984) Influence of tetracyclines on human polymorphonuclear leukocyte function. Antimicrob Agents Chemother 25:354
Golub LM, Suomalainen K, Sorsa T (1992) Host modulation with tetracyclines and their chemically modified analogues. Curr Opin Dent 2:80
Gradishar WJ (1997) An overview of clinical trials involving inhibitors of angiogenesis and their mechanisms of action. Invest New Drugs 15:49
Green DR, Kroemer G (2004) The pathophysiology of mitochondrial cell death. Science 305:626
Guerin C, Laterra J, Masnyk T, Golub LM, Brem H (1992) Selective endothelial growth inhibition by tetracyclines that inhibit collagenase. Biochem Biophys Res Commun 188:740
Gurubhagavatula I, Amarani Y, Pratico D, Ruberg FL, Albelda SM, Panetteri RA Jr (1998) Engagement of human PECAM-1 (CD31) on human endothelial cells increases intracellular calcium ion concentration and stimulates prostacyclin release. J Clin Invest 101:212
Hanemaaijer R, Visser H, Koolwijk P, Sorsa T, Salo T, Golub LM, van Hinsbergh VW (1998) Inhibition of MMP synthesis by doxycycline and chemically modified tetracyclines (CMTs) in human endothelial cells. Adv Dent Res 12:114
Hatton M, Day S, Southward S, DeReske M, Ross B, Seidlitz E, Singh G, Richardson M (2001) Metabolism of rabbit angiostatin glycoforms I and II in rabbits: angiostatin-I leaves the intravascular space faster and appears to have greater anti-angiogenic activity than angiostatin-II. J Lab Clin Med 138:83
Ingber D, Folkman J (1988) Inhibition of angiogenesis through modulation of collagen metabolism. Lab Invest 59:44
Islam MM, Franco CD, Courtman DW, Bendeck MP (2003) A nonantibiotic chemically modified tetracycline (CMT-3) inhibits intimal thickening. Am J Pathol 163:1557
Jiang L, Vivekanand J, Dhanabal M, Sukhatme VP, Alper SL (2001) Intracellular Ca2+ signaling in endothelial cells by the angiogenesis inhibitors endostatin and angiostatin. Am J Physiol Cell Physiol 280:C1140
Johnson MD, Kim HR, Chesler L, Tsao-Wu G, Bouck N, Polverini P (1994) Inhibition of angiogenesis by tissue inhibitor of matrix metalloproteinases. J Cell Physiol 160:194
Kalas W, Gilpin S, Yu JL, May L, Krchnakova H, Bornstein P, Rak J (2003) Restoration of thrombospondin 1 expression in tumor cells harbouring mutant ras oncogene by treatment with low doses of doxycycline. Biochem Biophys Res Commun 310:109
Kawasaki J, Davis GE, Davis MJ (2004) Regulation of Ca2+-dependent K+ current by alphavbeta3 integrin engagement in vascular endothelium. J Biol Chem 279:12959
Lamparter S, Slight SH, Weber KT (2002) Doxycycline and tissue repair in rats. J Lab Clin Med 139:295
Lee CZ, Hashimoto T, McCulloch CE, Yang GY, Young WL (2004) Doxycycline suppresses cerebral matrix metalloproteinase-9 and angiogenesis induced by focal hyperstimulation of vascular endothelial growth factor in a mouse model. Stroke 35:1715
Liekens S, De Clercq E, Neyts J (2001) Angiogenesis: regulators and clinical applications. Biochem Pharmacol 61:253
Mandal M, Das S, Chakraborti T, Mandal A, Chakraborti S (2003) Role of matrix metalloprotease-2 in oxidant activation of Ca2+ ATPase by hydrogen peroxide in pulmonary vascular smooth muscle plasma membrane. J Biosci 28:205
McMullen M, Keller R, Sussman M, Pumiglia K (2004) Vascular endothelial growth factor-mediated activation of p38 is dependent upon Src and RAFTK/Pyk2. Oncogene 23:1275
Missirlis E, Karakiulakis G, Maragoudakis ME (1990) Angiogenesis is associated with collagenous protein synthesis and degradation in the chick chorioallantoic membrane. Tissue Cell 22:419
Parangi S, O’Reilly MS, Christofori G, Holmgren L, Grosfeld J, Folkman J, Hanahan D (1996) Antiangiogenic therapy of transgenic mice impairs de novo tumor growth. Proc Natl Acad Sci U S A 93:2002
Pozzi A, LeVine WF, Gardner HA (2002) Low levels of matrix metalloproteinases 9 permit increased tumor angiogenesis. Oncogene 21:272
Rawal SY, Rawal YB (2001) Non-antimicrobial properties of tetracyclines—dental and medical implications. West Indian Med J 50:105
Reijerkerk A, Voest EE, Gebbink MFBG (2000) No grip, no growth: the conceptual basis of excessive proteolysis in the treatment of cancer. Eur J Cancer 36:1705
Ribatti D, Alessandri G, Vacca A, Iurlaro M, Ponzoni M (1998) Human neuroblastoma cells produce extracellular matrix-degrading enzymes, induce endothelial cell proliferation and are angiogenic in vivo. Int J Cancer 77:449
Ribatti D, Nico B, Vacca A, Iurlaro M, Roncali L (1999) Temporal expression of the matrix metalloproteinase MMP-2 correlates with fibronectin immunoreactivity during the development of the vascular system in the chick embryo chorioallantoic membrane. J Anat 195:39
Richardson M, Gunawan J, Hatton MWC, Seidlitz E, Hirte HW, Singh G (2002) Malignant ascites fluid (MAF), including ovarian-cancer-associated MAF, contains angiostatin and other factor(s) which inhibit angiogenesis. Gynecol Oncol 86:279
Richardson M, Singh G (2003) Observations on the use of the avian chorioallantoic membrane (CAM) model in investigations into angiogenesis. Curr Drug Targets Cardiovasc Hematol Disord 3:155
Rubins JB, Charboneau D, Alter MD, Bitterman PB, Kratzke RA (2001) Inhibition of mesothelioma cell growth in vitro by doxycycline. J Lab Clin Med 138:101
Saikali Z, Singh G (2003) Doxycycline and other tetracyclines in the treatment of bone metastasis. Anticancer Drugs 14:773
Seidlitz E, Korbie D, Marien L, Richardson M, Singh G (2004) Quantification of anti-angiogenesis using the capillaries of the chick chorioallantoic membrane demonstrates that the effect of human angiostatin is age-dependent. Microvasc Res 67:105
Sipos EP, Tamargo RJ, Weingart JD, Brem H (1994) Inhibition of tumor angiogenesis. Ann N Y Acad Sci 732:263
Slawomir MW, Dickson RB, Hawkins MJ (1997) Matrix metalloproteinase inhibitors. Invest New Drugs 15:61
Sorsa T, Konttinen YT, Lindy O, Suomalainen K, Ingman T, Saari H, Halinen S, Lee HM, Golub LM, Hall J (1993) Doxycycline protects serum alpha-1-antitrypsin from human neutrophil collagenase. Agents Actions Suppl 39:225
Suc I, Escargueil-Blanc I, Troly M, Salvayre R, Nègre-Salvayre A (1997) HDH and ApoA prevent cell death of endothelial cells induced by oxidized LDL. Arterioscler Thromb Vasc Biol 17:2158
Tamargo RJ, Bok RA, Brem H (1991) Angiogenesis inhibition by minocycline. Cancer Res 51:672
Warren MC, Bump EA, Medeiros D, Braunhut SJ (2000) Oxidative stress-induced apoptosis of endothelial cells. Free Radic Biol Med 29:537
Wijelath ES, Murray J, Rahman S, Patel Y, Ishida A, Strand K, Aziz S, Cardona C, Hammond WP, Savidge GF, Rafii S, Sobel M (2002) Novel vascular endothelial growth factor binding domains of fibronectin enhance vascular growth factor biological activity. Circ Res 91:25
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This work was supported by a Grant from the Cancer Research Society, Montreal, Canada
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Richardson, M., Wong, D., Lacroix, S. et al. Inhibition by doxycycline of angiogenesis in the chicken chorioallantoic membrane (CAM). Cancer Chemother Pharmacol 56, 1–9 (2005). https://doi.org/10.1007/s00280-004-0955-2
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DOI: https://doi.org/10.1007/s00280-004-0955-2