[Indomethacin induces apoptosis of K562 cells through activation of caspases and elevation of intracellular free calcium].
OBJECTIVE
To investigate the indomethacin (IN) induced apoptosis of K562 cells: (1) the expression, activation and subcellular localization of caspase-3 and -8; (2) the change of intracellular free calcium concentration ([fCa(2+)]i) and its mechanism; and (3) whether the cell apoptosis is cyclooxygenase (cox) dependent or not.


METHODS
Changes and subcellular localization of caspase-3 and -8 were observed by laser scanning confocal microscopy (LSCM); expression and activation of caspase-3 and -8 proteins by Western blotting; changes of intracellular [fCa(2+)]i by LSCM coupling with a calcium-fluorescence probe and calcium chelator EGTA blocking test; and cox inhibitor effect by MTT assay.


RESULTS
(1) LSCM assay and Western blotting showed that caspase-3 and -8 localized in cytoplasm and nucleus dispersedly and spottedly and were upregulated with the increasing of IN doses. Western blotting also showed the cleavage and activation of caspase-3 and -8 during IN-induced apoptosis. (2) The increase of [fCa(2+)]i in K562 cells was parallel to the increase of IN concentration regardless of the presence or absence of EGTA. But with EGTA treatment, [fCa(2+)]i was much less than that without EGTA treatment. (3) Low dose of IN or other cox inhibitors could not exert cytotoxic effects on K562 cells whereas high dose of IN could.


CONCLUSIONS
(1) The upregulation and activation of caspase-3 and -8 play a fundamental role in apoptosis induced by IN in K562 cells. Both cytoplasm and nucleus are locations where caspase-3 and -8 localized. (2) The increase of [fCa(2+)]i may be critical in the modulation of apoptosis; the extracellular calcium influx is the main source of the elevation of [fCa(2+)]i in K562 cells and can be blocked by the calcium chelator EGTA;the release of calcium from intracellular calcium store is also an important source of the intracellular calcium which can trigger apoptosis without the extracellular calcium influx. (3) Apoptosis of K562 cells induced by IN is cox-independent.
Zhou
G
G
Department of Hematology, The Second Affiliated Hospital of Hunan Medical University, Changsha 410011, China.
Zhang
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G
D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism*; D053148:Caspase 3 / Q000378:metabolism*; D053181:Caspase 8 / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D007213:Indomethacin / Q000494:pharmacology*; D020014:K562 Cells; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism
D011451:Prostaglandin-Endoperoxide Synthases; D053148:Caspase 3; D053181:Caspase 8; D002118:Calcium; D007213:Indomethacin