Antioxidative action of N-a-tosyl-L-lysine chloromethyl ketone prevents death of glutathione-depleted cardiomyocytes induced by hydrogen peroxide
Kiyoshi Takahashi, Kyohei Oyama, Hironori Motoshige, Koichi Sakurai
DOI: 10.4236/jbpc.2010.13019   PDF   HTML   XML   4,879 Downloads   9,238 Views   Citations


Hydrogen peroxide (H2O2) induces the hypertrophy in cultured H9c2 cardiomyocytes and cell death in glutathione (GSH)-depleted H9c2 cells. In the present study, we observed that pretreatment with a serine protease inhibitor, N-a-tosyl-L-lysine chloromethyl ketone (TLCK), significantly prevented the H2O2-induced cell damages in GSH-depleted H9c2 cells in a concentration-dependent manner. The phase contrast microscopy revealed that although the exposure of the GSH-depleted H9c2 cells to H2O2 resulted in a globular shape of the cells, TLCK prevented the occurrence of H2O2-induced morphological changes. TLCK also inhibited the generation of reactive oxygen species in the cells after addition of H2O2, suggesting that the antioxidant action of TLCK is involved in the protection against the cell damages by H2O2. Application of TLCK after ~30 min of exposure to H2O2 could significantly protect the cells from cell damages. The other serine protease inhibitors that were tested could not prevent the cell damages in GSH- depleted H9c2 cells. Pretreatment with an inhibitor of nuclear factor-kB translocation into the nucleus and a proteasome inhibitor did not prevent the cell damages in GSH-depleted H9c2 cells. An inhibitor of p53 significantly prevented the cell damages in GSH-depleted H9c2 cells. These results suggest that antioxidative action of TLCK prevents the death of GSH-depleted H9c2 cardiomyocytes induced by H2O2.

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Takahashi, K. , Oyama, K. , Motoshige, H. and Sakurai, K. (2010) Antioxidative action of N-a-tosyl-L-lysine chloromethyl ketone prevents death of glutathione-depleted cardiomyocytes induced by hydrogen peroxide. Journal of Biophysical Chemistry, 1, 164-171. doi: 10.4236/jbpc.2010.13019.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Takano, H., Zou, Y., Hasegawa, H., Akazawa, H., Nagai, T. and Komuro, I. (2003) Oxidative stress-induced signal transduction pathways in cardiac myocytes: Involvement of ROS in heart diseases. Antioxidants & Redox Signaling, 5, 789-794.
[2] Trachootham, D., Lu, W., Ogasawara, M.A., Nilsa, R.D. and Huang, P. (2008) Redox regulation of cell survival. Antioxidants & Redox Signaling, 10, 1343-1374.
[3] Chen, Q.M., Tu, V.C., Wu, Y. and Bahl, J.J. (2000) Hydrogen peroxide dose dependent induction of cell death or hypertrophy in cardiomyocytes. Archives of Biochemistry and Biophysics, 373, 242-248.
[4] Tanaka, H., Sakurai, K., Takahashi, K. and Fujimoto, Y. (2003) Requirement of intracellular free thiols for hydrogen peroxide-induced hypertrophy in cardiomyocytes. Journal of Cellular Biochemistry, 89, 944-955.
[5] Arrigo, A.P. (1999) Gene expression and the thiol redox state. Free Radical Biology & Medicine, 27, 936-944.
[6] Carmel-Harel, O. and Storz, G. (2000) Roles of the glutathione- and thioredoxin-dependent reduction systems in the Escherichia coli and Saccharomyces cerevisiae responses to oxidative stress. Annual Review of Microbiology, 54, 439-461.
[7] Filomeni, G., Rotilio, G. and Ciriolo, M.R. (2002) Cell signaling and the glutathione redox system. Biochemical Pharmacology, 64, 1057-1064.
[8] Shukla, G.S., Shukla, A., Potts, R.J., Osier, M., Hart, B.A. and Chiu, J.F. (2000) Cadmium-mediated oxidative stress in alveolar epithelial cells induces the expression of gamma-glutamylcysteine synthetase catalytic subunit and glutathione S-transferase alpha and pi isoforms: potential role of activator protein-1. Cell Biology and Toxicology, 16, 347-362.
[9] Solary, E., Eymin, B., Droin, N. and Haugg, M. (1998) Proteases, proteolysis, and apoptosis. Cell Biology and Toxicology, 14, 121-132.
[10] Stenson-Cox, C., FitzGerald, U. and Samali, A. (2003) In the cut and thrust of apoptosis, serine proteases come of age. Biochemical Pharmacology, 66, 1469-1474.
[11] Kim, H., Lee, H.S., Chang, K.T., Ko, T.H., Beak, K.J. and Kwon, N.S. (1995) Chloromethyl ketones block induction of nitric oxide synthase in murine macrophages by preventing activation of nuclear factor-kappa B. The Journal of Immunology, 154, 4741-4748.
[12] Abate, A. and Schr?der, H. (1998) Protease inhibitors protect macrophages from lipopolysaccharide-induced cytotoxicity: Possible role for NF-?B. Life Science, 62, 1081-1088.
[13] Gong, B., Chen, Q., Endlich, B., Mazumder, S. and Almasan, A. (1999) Ionizing radiation-induced, Bax-mediated cell death is dependent on activation of cysteine and serine proteases. Cell Growth & Differentiation, 10, 491- 502.
[14] Rideout, H.J., Zang, E., Yeasmin, M., Gordon, R., Jabado, O., Park, D.S. and Stefanis, L. (2001) Inhibitors of trypsin- like serine proteases prevent DNA damage-induced neuronal death by acting upstream of the mitochondrial checkpoint and of p53 induction. Neuroscience, 107, 339-352.
[15] Adams, J. and Stein, R. (1996) Novel inhibitors of the proteasome and their therapeutic use in inflammation. Annual Reports in Medicinal Chemistry, 31, 279-288.
[16] Grisham, M.B., Palombella, V.J., Elliott, P.J., Conner, E.M., Brand, S., Wong, H.L., et al. (1999) Inhibition of NF-?B activation in vitro and in vivo: Role of 26S proteasome. Methods in Enzymology, 300, 345-363.
[17] Chen, K., Albano, A., Ho, A. and Keaney, J.F.Jr. (2003) Activation of p53 by oxidative stress involves platelet-derived growth factor-? receptor-mediated ataxia telangiectasia mutated (ATM) kinase activation. The Journal of Biological Chemistry, 278, 39527-39533.
[18] Setsukinai, K., Urano, Y., Kakinuma, K., Majima, H.J. and Nagano, T. (2003) Development of novel fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species. The Journal of Biological Chemistry, 278, 3170-3175.
[19] Gao, D., Sakurai, K., Katoh, M., Chen, J. and Ogiso, T. (1996) Inhibition of microsomal lipid peroxidation by baicalein: A possible formation of an iron-baicalein complex. Biochemistry and Molecular Biology International, 39, 215-225.
[20] Myhre, O., Andersen, J.M., Aarnes, H. and Fonnum, F. (2003) Evaluation of the probes 2',7'-dichlorofluorescin diacetate, luminol, and lucigenin as indicators of reactive species formation. Biochemical Pharmacology, 65, 1575- 1582.
[21] Stone, J.R. and Yang, S. (2006) Hydrogen peroxide: A signal messenger. Antioxidants & Redox Signaling, 8, 243-270.
[22] Lin, Y.Z., Yao, S.Y., Veach, R.A., Torgerson, T.R. and Hawiger, J. (1995) Inhibition of nuclear translocation of transcription factor NF-?B by a synthetic peptide containing a cell membrane-permeable motif and nuclear localization sequence. The Journal of Biological Chemistry, 270, 14255-14258.
[23] Komarov, P.G., Komarova, E.A., Kondratov, R.V., Christov-Tselkov, K., Coon, J.S., Chernov, M.V., et al. (1999) A chemical inhibitor of p53 that protects mice from the side effects of cancer therapy. Science, 285, 1733-1737.
[24] Burns, T.F. and El-Deiry, W.S. (1999) The p53 pathway and apoptosis. Journal of Cellular Physiology, 181, 231- 239.
[25] Chen, S.J., Wang, J.L., Chen, J.H. and Huang, R.N. (2002) Possible involvement of glutathione and p53 in trichloroethylene- and perchloroethylene-induced lipid peroxidation and apoptosis in human lung cancer cells. Free Radical Biology & Medicine, 33, 464-472.
[26] Renzing, J., Hansen, S. and Lane, D.P. (1996) Oxidative stress is involved in the UV activation of p53. Journal of Cell Science, 109, 1105-1112.
[27] Henkel, T., Machleidt, T., Alkalay, I., Kr?nke, M., Ben- Neriah, Y. and Baeuerle, P.A. (1993) Rapid proteolysis of I?B-? is necessary for activation of transcription factor NF-?B. Nature, 365, 182-185.
[28] Bai, J. and Cederbaum, A.I. (2006) Overexpression of CYP2E1 in mitochondria sensitizes HepG2 cells to the toxicity caused by depletion of glutathione. The Journal of Biological Chemistry, 281, 5128-5136.

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