Upregulation and activation of caspase-3 or caspase-8 and elevation of intracellular free calcium mediated apoptosis of indomethacin-induced K562 cells

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Journal Title:
CHINESE MEDICAL JOURNAL
Issue:
Volume , Issue 07, 2004
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Key Word:
Indomethacin;K562 cells;Apoptosis;Caspase;calcium signal

Abstract: Background A nonsteroidal anti-inflammatory drug, indomethacin, has been shown to have anti-leukemic activity and induce leukemic cell opoptosis. This study was to elucidate the mechanism of indomethacin-induced K562 cell apoptosis.Methods K562 cells were grown in RPMI 1640 medium and treated with different doses of indomethacin (0 μmol/L, 100 μmol/L, 200 μmol/L, 400 μmol/L, 800 μmol/L) for 72 hours. The cells were harvested, and cell viability or apoptosis was analyzed using MTT assay and AO/EB stain, combining laser scanning confocal microscopy (LSCM) technique separately. For the localization and distribution of intracellular caspase-3 or caspase-8 protein, immunofluorescence assay was carried out. To reveal the activation of caspase-3 or caspase-8 in indomethacin-treated cells, Western blot detection was used. The change in intracellular free calcium was determined by Fluo-3/ Am probe labeling combined with LSCM. Results Indomethacin could lead to K562 cell apoptosis and inhibit cell viability in a concentration-dependent manner. An increased expression of intracellular caspase-3 or caspase-8 was observed at higher doses of indomethacin (400-800 μmol/L). Western blot results showed upregrulation and activation in both caspase-3 and caspase-8 protein. Under indomethacin intervention, the levels of intracellular free calcium showed a significant increase. Blocking the activity of cyclooxygenase did not abolish the effects of indomethacin on K562 cell apoptosis.Conclusions Activation and upregulation of caspase-3 or caspase-8 protein were responsible for Indomethacin-induced K562 cell apoptosis. Variation of intracellular free calcium might switch on the apoptotic pathway and the proapoptotic effect of indomethacin might be cyclooxygenase-independent.

  • [1]Cortez D, Kadlec L, Pendergast AM: Structural and signaling requirements for BCR-ABL-mediated transformation and inhibition of apoptosis. Mol Cell Biol 1995;15:5531-5541.
  • [2]Dubrez L, Eymin B, Sordet O, et al. BCR-ABL delays apoptosis upstream of procaspase-3 activation. Blood 1998;91:2415-2422.
  • [3]Thornberry NA, Lazebnik Y: Caspases: enemies within. Science 1998;281:1312-1316.
  • [4]Villa P, Kaufman SH, Earnshaw WC: Caspases and caspases inhibitors. TIBS 1997;22:388-393.
  • [5]Wang HG, Pathan N, Ethell IM, et al. Ca2+-induced apoptosis through calcineurin dephosphorylation of BAD. Science 1999;284:339-343.
  • [6]Cryer B, Feldman: Cyclooxygenase-1 and cyclooxygenase-2 selectivity of widely used nonsteroidal anti-inflammatory drugs. Am J Med 1998;104:413-421.
  • [7]Zhang GS, Tu CQ, Zhang GY, et al. Indomethacin induces apoptosis and inhibits proliferation in chronic myeloid leukemia cells. Leukemia Res 2000;24:385-392.
  • [8]Elder DJ, Hague A, Hicks DJ, et al. Differential growth inhibition by the aspirin metabolite salicylate in human colorectal tumor cell lines: enhanced apoptosis in carcinoma and in vitro-transformed adenoma relative to adenoma cell lines. Cancer Res 1996;56:2273-2276.
  • [9]Klampfer L, Cammenga J, Wisniewski HG, et al. Sodium salicylate activates caspases and induces apoptosis of myeloid leukemia cell lines. Blood 1999;93:2386-2394.
  • [10]Vordra ce KJ, Stika J, Soucek K, et al. Inhibitors of arachidonic acid metabolism potentiate tumour necrosis factor-α-induced apoptosis in HL-60 cells. Eur J Pharmacol 2001;424;1-11.
  • [11]Bellosillo B, Pique M, Barragan M, et al. Aspirin and salicylate induce apoptosis and activation of caspase in B-cell chronic lymphocytic leukemia cells. Blood 1998;92:1406-1414.
  • [12]Hanif R, Pittas A, Feng Y, et al. Effects of nonsteroidal antiinflammatory drugs on proliferation and on induction of apoptosis in colon cancer cells by a prostaglandin- independent pathway. Biochem Pharmacol 1996;52:245-273.
  • [13]Dai J, Weinberg RS, Waxman S, et al. Malignant cells can be sensitized to undergo growth inhibition and apoptosis by arsenic trioxide through modulation of the glutathione redox syotem. Blood 1999;93:268-277.
  • [14]Khan NA, Meyniel JP, Deschaux P. Ca2+/calmodulin and protein kinase C regulation of serotomin transport in human K562 lymphocytes. Cellular Immunol 1996;172:269-274.
  • [15]Gavioli D, Spisani S, Giuliani AL, et al. Dual mechanism in induction of human neutrophil cytotoxicity: activation of protein kinase C and elevation in intracellular calcium. Clin Exp Immunol 1990;80:247-251.
  • [16]Kimura K, Sasano H, Shimosegawa T, et al. Ultrastructural and confocal laser scanning microscopies examination of TUNEL-positive cells. J Pathol 1997;181:235-242.
  • [17]McGahon A, Bissonnette R, Schmitt M, et al. Bcr-abl maintains resistance of chronic myelogenous leukemia cells to apoptotic cell death. Blood 1994;83:1179-1187.
  • [18]Martins LM, Mesner PW, Kottke TJ, et al. Comparison of caspase activation and subcellular localization in HL-60 and K562 cells undergoing etoposide-induced apoptosis. Blood 1997;90:4283-4296.
  • [19]Lindqvist C, Holmberg C, Detken C. Rapid Ca2+ mobilization in single LGL cells upon interaction with K562 target cells-role of the CD18 and CD16 molecules. Cell Immunol 1995;165:71-76.
  • [20]Rogue PJ, Malviya AN: Calcium signals in the cell nucleus. Strasbourg, France, August 20-23, 1998. EMBO J 1999;18:5147-5152.
  • [21]Waskewich C, Blumenthal RD, Li HL, et al. Celecoxib exhibits the greatest potency amongst cyclooxygenase (COX) inhibitors for growth inhibition of COX-2 negative hematopoietic and epithelial cell line. Cancer Res 2002;62:2029-2033.
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