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Feng XQ, You Y, Xiao J, Zou P.
Thapsigargin-induced apoptosis of K562 cells
and its mechanism
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2006 Feb;14(1):25-30.
The aim was to study the apoptotic induction effect of thapsigargin on
leukemia cell line K562 and its possible mechanism. After the treatment of
leukemia cell line K562 by thapsigargin, morphological change of apoptotic
cells was investigated by AO/EB fluorescent staining under fluorescent
microscope; apoptosis rate was determined with annexin V-FITC/PI double
staining by flow cytometry; intracellular calcium concentrations ([Ca(2+)]i)
were measured by fluorescence spectrophotometer with calcium sensitive
fluorescence indicator Fura-2/AM; mitochondrial transmembrance potentials
(Delta Psi m) was detected on flow cytometry through staining of Rhodamine
(Rh123); the changes of caspase-3, -7, -9, -12, cytochrome C, GRP78 proteins
were detected by Western blot. The results showed that K562 cells cultured
in 4 micromol/L thapsigargin for 48 hours exhibited typical morphological
changes of apoptotic cells under fluorescent microscope, including shrinkage
of cell, condensation of chromatin, breakage of nuclear, formation of
apoptotic bodies, fluorescence of yellow green and pellet observed in early
apoptoyic cells and hyacinth fluorescence of chromatin showed in late
apoptotic cells. 24 and 48 hours after exposure to 1, 2, 4, 8 micromol/L
thapsigargin, the apoptotic rates of K562 were respectively 7.51%, 11.65%,
23.22%, 30.56% and 12.85%, 20.27%, 31.51%, 44.16%, in dose-dependent manner,
and were statistically significant when compared with the controls (P <
0.05). The apoptotic rate of K562 was dose- and time-dependent in experiment
range. The enhancement of [Ca(2+)]i and the decrease of the Delta Psi m in
K562 cells were induced by thapsigargin and were dose-dependent in
experiment range, compared with control, P < 0.05. Western blot results
indicated that cleavage and activation of caspase-3, -7, -9, -12, releasing
of cytochrome C from mitochondria, upregulation of GRP78 expression at the
endoplasmic reticulum were induced in K562 cells after 24 hours exposure of
4 micromol/L thapsigargin. It is concluded that thapsigargin induces
endoplasmic reticulum stress-induced apoptosis in K562 cells. Endoplasmic
reticulum is a novel important initiatory site of apoptosis in cells; the
cleavage and activation of caspase-3, -7, -9, -12 play very important role
in endoplasmic reticulum stress-induced apoptosis of K562 cells and is one
of the important mechanisms for thapsigargin-induced apoptosis.
Thapsigargin-induced apoptosis in K562 cells is associated closely with the
disruption of the Delta Psi m and the release of cytochrome C from
mitochondria, mitochondria participates in endoplasmic reticulum
stress-induced apoptosis in K562 cells.
PMID: 16584585
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