Upregulation and activation of caspase-3 or caspase-8 and elevation of intracellular free calcium mediated apoptosis of indomethacin-induced K562 cells.
BACKGROUND
A nonsteroidal anti-inflammatory drug, indomethacin, has been shown to have anti-leukemic activity and induce leukemic cell apoptosis. 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 micromol/L, 100 micromol/L, 200 micromol/L, 400 micromol/L, 800 micromol/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 micromol/L). Western blot results showed upregulation 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.
Zhang
Guang-sen
GS
Institute of Molecular Hematology/Division of Hematology, Second Xiang-Ya Hospital, Central South University, Changsha, Hunan 410011, China. zgsllzy@public.cs.hn.cn
Zhou
Guang-biao
GB
Dai
Chong-wen
CW
D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism*; D053148:Caspase 3; D053181:Caspase 8; D020169:Caspases / Q000235:genetics* / Q000378:metabolism; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D006801:Humans; D007213:Indomethacin / Q000494:pharmacology*; D020014:K562 Cells
D016861:Cyclooxygenase Inhibitors; C505407:CASP3 protein, human; C505530:CASP8 protein, human; D053148:Caspase 3; D053181:Caspase 8; D020169:Caspases; D002118:Calcium; D007213:Indomethacin