Cell and Tissue Biology

, Volume 3, Issue 1, pp 56–60 | Cite as

Activity of matrix metalloproteinases in normal and transformed mouse fibroblasts exposed to antioxidants

  • I. V. Voronkina
  • K. M. Kirpichnikova
  • L. V. Smagina
  • I. A. GamaleyEmail author


The effects of two antioxidants on the activity of matrix metalloproteinases (MMP) secreted by normal (3T3) and transformed (3T3-SV40) mouse fibroblasts were examined. We compared the action of N-acetylcysteine (NAC) and alpha-lipoic acid (ALA) on two gelatinases, MMP-2 and MMP-9. Gel zymography demonstrated that activity of MMP-2 was higher in normal 3T3 cells, whereas, in transformed 3T3-SV40 cells, the MMP-9 activity was higher. NAC treatment for 2–6 h completely suppressed MMP-2 and MMP-9 activity in both cell lines. The inhibitory effect did not depend on NAC concentration within the range of 1–10 mM. ALA (1.2 mM) did not affect the cells very dramatically; it decreased the MMP-2 activity in both types of cells. MMP-9 activity in the presence of ALA was decreased in 3T3 cells and slightly increased in 3T3-SV40 cells. The activity of the membrane bound and intracellular MMP was not changed under the same conditions. In conclusion, the altered activity of MMP in the presence of antioxidant may influence the intracellular signaling and cell functions.

Key words

transformed fibroblasts matrix metalloproteinases N-acetylcystein alpha-lipoic acid 



matrix metalloproteinase


alpha-lipoic acid


reduced glutathione




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  1. Björklund, M. and Koivunen, E., Gelatinase-Mediated Migration and Invasion of Cancer Cells, Biochim. biophys. acta., 2005, vol. 1755, pp. 37–69.PubMedGoogle Scholar
  2. Bradford, M.M., A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding, Anal. Biochem., 1976, vol. 72, pp. 248–254.PubMedCrossRefGoogle Scholar
  3. Filatova N. A., Kirpichnikova K.M, Gamaley I.A. Reorganization of Actin Cytoskeleton in 3T3-SV40 Cells and their Sensitivity to Lysis by Natural Killer Cells, Cell Tis. Biol., 2008, vol. 2, no. 2, pp. 261–267.Google Scholar
  4. Filatova, N.A., Kirpichnikova, K.M., Gamaley, I.A., N-Acetylcysteine Reduces Transformed 3T3-SV40 Fibroblast Sensitivity to Lysis by Natural Killer Cells, Tsitologiia, 2006, vol. 48, no. 5, pp. 438–442.PubMedGoogle Scholar
  5. Gamaley, I.A., Aksenov, N.D., Efremova, T.N., and Kirpichnikova, K.M., Effect of Agents Changing the Intracellular Level of Reactive Oxygen Species on the Cell Cycle Phase Distribution in 3T3 and 3T3SV40 Cell Lines, Tsitologiia, 2003, vol. 45, pp. 26–33.Google Scholar
  6. Gamaley, I., Efremova, T., Kirpichnikova, K., Kever, L., Komissarchik, Y., Polozov, Yu., and Khaitlina, T., N-Acetylcysteine-Induced Changes in Susceptibility of Transformed Eukaryotic Cells to Bacterial Invasion, Cell Biol. Int., 2006, vol. 30, pp. 319–325.PubMedCrossRefGoogle Scholar
  7. Goldman, S., Weiss, A., Eyali, V., and Shalev, E., Differential Activity of the Gelatinases (Matrix Metalloproteinases 2 and 9) in the Fetal Membranes and Decidua, Associated with Labour, Mol. Hum. Reprod., 2003, vol. 9, pp. 367–373.PubMedCrossRefGoogle Scholar
  8. Hwang, E.-S. and Lee, H.J., Allyl Isothiocyanate and Its NAcetylcysteine Conjugate Suppress Metastasis via Inhibition of Invasion, Migration, and Matrix Metalloproteinase-2/-9 Activities in SK-Hep 1 Human Hepatoma Cells, Exp. Biol. Med., 2006, vol. 231, pp. 421–430.Google Scholar
  9. Kawakami, S., Kageyama, Y., Fujii, Y., Kihara, K., and Oshima, H., Inhibitory Effect of N-Acetylcysteine on Invasion and MMP-9 Production of T24 Human Bladder Cancer Cells, Anticancer Res., 2001, vol. 21, pp. 213–219.PubMedGoogle Scholar
  10. Klisho, E.V., Kondakova, I.V., Choinzonov, E.L., and Vasil’eva, O.S., Prognostic Significance of Proteases in Patients with Squamous Cell Carcinomas of the Head and Neck, Byull. Sib. Div. Russ. Acad. Med. Sci., 2005, vol. 116, pp. 82–91.Google Scholar
  11. Laemmli, U.K., Cleavage of Structural Proteins During the Assembly of the Head of Bacteriophage T4, Nature, 1970, vol. 227, pp. 680–683.PubMedCrossRefGoogle Scholar
  12. Lai, C.F., Seshadri, V., Huang, K., Shao, J.S., Cai, J., Vattikuti, R., Schumacher, A., Loewy, A.P., Denhardt, D.T., Rittling, S.R., and Towler, D.A., An Osteopontin-NADPH Oxidase Signaling Cascade Promotes Pro-Matrix Metalloproteinase 9 Activation in Aortic Mesenchymal Cells, Circ. Res., 2006, vol. 98, pp. 1479–1489.PubMedCrossRefGoogle Scholar
  13. Moini, H., Packer, L., and Saris, N.E., Antioxidant and Prooxidant Activities of Alpha-Lipoic Acid and Dihydrolipoic Acid, Toxicol. Appl. Pharmacol., 2002, vol. 182, pp. 84–90.PubMedCrossRefGoogle Scholar
  14. Mott, J.D. and Werb, Z., Regulation of Matrix Biology by Matrix Metalloproteinases, Cur. Opinion Cell Biol., 2004, vol. 16, pp. 558–564.CrossRefGoogle Scholar
  15. Okamoto, T., Akaike, T., Sawa, T., Miyamoto, Y., van der Vliet, A., and Maeda, H., Activation of Matrix Metalloproteinases by Peroxynitrite-Induced Protein S-Glutathiolation via Disulfide S-Oxide Formation, Biol. Chem., 2001, vol. 27, pp. 29596–29602.Google Scholar
  16. Oliver, G.W., Stettler-Stevenson, W.G., and Kleiner, D.E., Zymography, Casein Zymography and Reverse Zymography: Activity Assays for Proteases and Their Inhibitors, in Handbook of Proteolyitic Enzymes, San Diego: Academic, 1999, pp. 61–76.Google Scholar
  17. Packer, L., Witt, E.H., and Tritschler, H.J., Alpha-Lipoic Acid as a Biological Antioxidant, Free Radic Biol.Med., 1995, vol 19, pp. 227–250.PubMedCrossRefGoogle Scholar
  18. Pei, P., Horan, M.P., Hille, R., Hemann, C.F., Schwendeman, S.P., and Mallery, S.R., Reduced Nonprotein Thiols Inhibit Activation and Function of MMP-9: Implications for Chemoprevention, Free Rad. Biol. Med., 2006, vol. 41, pp. 1315–1324.PubMedCrossRefGoogle Scholar
  19. Schnaeker, E.-M., Ossig, R., Ludwig, T., Dreier, R., Oberleithner, H., Wilhelmi, M., and Schneider, S.W., Microtubule-Dependent Matrix Metalloproteinase-2/Matrix Metalloproteinase-9 Exocytosis: Prerequisite in Human Melanoma Cell Invasion. Cancer Res., 2004, 64, pp. 8924–8931.PubMedCrossRefGoogle Scholar
  20. Sen, C.K. and Packer, L., Thiol Homeostasis and Supplements in Physical Exercise, Am. J. Clin. Nutr., 2000, vol. 72, no. 2, Suppl., pp. 653S–669S.PubMedGoogle Scholar
  21. Spingman, E.B., Angleton, E.L., Birkedal-Hansen, H., and Van Wart, H.E., Multiple Modes of Activation of Latent Human Fibroblast Collagenase: Evidence for the Role of a Cys73 Active-Site Zinc Complex in Latency and a “Cysteine Switch” Mechanism for Activation, Proc. Natl. Acad. Sci. USA, 1990, vol. 87, pp. 364–368.CrossRefGoogle Scholar
  22. Spolarics, Z. and Wu, J.X., Role of Glutathione and Catalase in H2O2 Detoxification in LPS-Activated Hepatic Endothelial and Kupffer Cells, Am. J. Physiol., 1997, vol. 273, pp. G1304–G1311.PubMedGoogle Scholar
  23. Van Wart, H.E. and Birkedal-Hansen, H., The Cysteine Switch: a Principle of Regulation of Metalloproteinase Activity with Potential Applicability to the Entire Matrix Metalloproteinase Gene Family, Proc.Natl.Acad. Sci. USA, 1990, vol. 87, pp. 5578–5582.PubMedCrossRefGoogle Scholar
  24. Voronkina, I.V., Kharisov, A.M., Blinova, M.I., Paramonov, B.A., Potokin, I.L., and Pinaev, G.P., The “Air Pouch” Model in Mice and a Study of the Wound Fluid Proteolytic Activity, Tsitologiia, 2002, vol. 44, no. 3, pp. 270–276.PubMedGoogle Scholar
  25. Weiss, A., Goldman, S., Ben Shlomo, I., Eyali, V., Leibovitz, S., and Shalev, E., Mechanisms of Matrix Metalloproteinase-9 and Matrix Metalloproteinase-2 Inhibition by NAcetylcysteine in the Human Term Decidua and Fetal Membranes, Am. J. Obstet. Gynecol. 2003, vol. 189, pp. 1758–1763.PubMedCrossRefGoogle Scholar
  26. Westermarck, J. and Kahari, V., Regulation of Matrix Metalloproteinase Expression in Tumor Invasion, FASEB J., 1999, vol. 13, pp. 781–792.PubMedGoogle Scholar
  27. Zafarullah, M., Li, W.Q., Sylvester, J., and Ahmad, M., Molecular Mechanisms of N-Acetylcysteine Actions, CMLS Cell Mol. Life Sci., 2003, vol. 60, pp. 6–20.CrossRefGoogle Scholar
  28. Zhang, H.S. and Wang, S.Q., Salvianolic Acid B from Salvia miltiorrhiza Inhibits Tumor Necrosis Factor-Alpha (TNF-alpha)-Induced MMP-2 Upregulation in Human Aortic Smooth Muscle Cells via Suppression of NAD(P)H Oxidase-Derived Reactive Oxygen Species, J. Mol. Cell Cardiol., 2006, vol. 41, pp. 138–148.PubMedCrossRefGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2009

Authors and Affiliations

  • I. V. Voronkina
    • 1
  • K. M. Kirpichnikova
    • 1
  • L. V. Smagina
    • 1
  • I. A. Gamaley
    • 1
    Email author
  1. 1.Institute of Cytology Russian Academy of SciencesSt. PetersburgRussia

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