Feng Xian-Qi, You Yong, Xiao Juan, Zou Ping
Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
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.
目的是研究毒胡萝卜素对白血病细胞系K562的凋亡诱导作用及其可能机制。用毒胡萝卜素处理白血病细胞系K562后，在荧光显微镜下通过AO/EB荧光染色观察凋亡细胞的形态变化；采用annexin V-FITC/PI双染法通过流式细胞术测定凋亡率；用钙敏感荧光指示剂Fura-2/AM通过荧光分光光度计测量细胞内钙浓度（[Ca(2+)]i）；通过罗丹明（Rh123）染色在流式细胞仪上检测线粒体跨膜电位（ΔΨm）；用Western blot检测caspase-3、-7、-9、-12、细胞色素C、GRP78蛋白的变化。结果显示，在4 μmol/L毒胡萝卜素中培养48小时的K562细胞在荧光显微镜下呈现出典型的凋亡细胞形态变化，包括细胞皱缩、染色质凝聚、核破裂、凋亡小体形成，早期凋亡细胞呈现黄绿色荧光和细胞团块，晚期凋亡细胞染色质呈现蓝紫色荧光。在暴露于1、2、4、8 μmol/L毒胡萝卜素24和48小时后，K562细胞的凋亡率分别为7.51%、11.65%、23.22%、30.56%和12.85%、20.27%、31.51%、44.16%，呈剂量依赖性，与对照组相比具有统计学意义（P < 0.05）。在实验范围内，K562细胞的凋亡率呈剂量和时间依赖性。毒胡萝卜素诱导K562细胞内[Ca(2+)]i升高和ΔΨm降低，在实验范围内呈剂量依赖性，与对照组相比，P < 0.05。Western blot结果表明，在4 μmol/L毒胡萝卜素处理24小时后，K562细胞中caspase-3、-7、-9、-12被切割激活，细胞色素C从线粒体释放，内质网中GRP78表达上调。结论是毒胡萝卜素诱导K562细胞发生内质网应激诱导的凋亡。内质网是细胞凋亡新的重要起始位点；caspase-3、-7、-9、-12的切割和激活在K562细胞内质网应激诱导的凋亡中起非常重要的作用，是毒胡萝卜素诱导凋亡的重要机制之一。毒胡萝卜素诱导K562细胞凋亡与ΔΨm的破坏和细胞色素C从线粒体的释放密切相关，线粒体参与K562细胞内质网应激诱导的凋亡。
