Changes of Caspase-3 in Deferoxamine-Induced Apoptosis of HL-60 Cells_West China Medical Journal

Authors：

1. Department of Hematology, Zhengzhou Children′s Hospital, Zhengzhou, Henan 450003, P.R. China;2. Department of Pediatrics, West China Second Hospital, Sichuan University, Chengdu, Sichuan 610041, P.R. China;

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Keywords：

HL-60细胞; 去铁胺; 脱噬作用; caspase-3

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【摘要】目的 探讨铁螯合剂去铁胺（DFO）对诱导白血病细胞HL-60的分子机制。方法 2003年7-12月用钙黄绿素（calcein）检测HL-60细胞LIP。台盼蓝活细胞拒染实验进行活细胞计数及细胞存活率测定；光镜形态学观察及流式细胞仪（FCM）等方法检测HL-60细胞凋亡；比色法检测caspase-3（基于pNA标记底物的比色法）活性。结果 ①不同浓度的DFO作用于HL-60细胞后，随培养时间延长及DFO浓度的增加，动态铁池降低，细胞生存率逐渐下降，凋亡率增加，显示一定的时间剂量依赖性。②HL-60细胞在不同浓度的DFO作用下，caspase-3的活性逐渐升高。50、100 μmol/L DFO作用于HL-60细胞24 h，caspase-3酶活性升高明显，与对照组相比，有统计学意义（P lt;0.001）；相关分析结果显示，HL-60细胞LIP的改变与caspase-3活性变化呈负相关系（r=-0.887，P lt;0.05）。结论 DFO诱导白血病细胞凋亡的作用可能与螯合细胞内铁，降低细胞LIP，激活caspase-3，最终实施细胞凋亡密切相关。
【Abstract】 Objective To observe the changes of caspase-3 activity during apoptosis of HL-60 cells induced by an iron deferoxamine (DFO). Methods Exponentially growing HL-60 cells (1×106/mL) were used in this experiment from July 2003 to December 2003. The study groups were divided as follows: DFO group, iron+DFO group and control group. The viability was detected by typanblue, apoptosis was assessed by morphological study and flow cytometry (FCM) assay, and the caspase-3 activity was detected by melorimetry. The intracellular label iron pool (LIP) was measured with a fluorimetric assay using the metalsensitive probe calcein-AM. Results ①When HL-60 cells were incubated with different concentrations of DFO, viability assay was lower than that in the control group at the 12th, 24th and 48th hour (P lt;0.05). ② The cells incubated with different concentrations of DFO showed dose-time dependence and was much higher than that in the control group (P lt;0.01). ③The caspase-3 activity was significantly higher in the apoptotic cells than that in the control cells. Conclusions The apoptosis of HL-60 cells induced by DFO may be correlated with the decrease of cellular LIP and activity of caspase-3.

Citation： JIA Guocun,LI Fengyi,GAO Ju. Changes of Caspase-3 in Deferoxamine-Induced Apoptosis of HL-60 Cells. West China Medical Journal, 2010, 25(9): 1683-1685. doi: Copy

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1. Meggiato T, Calabrese F, De Cesare CM, et al. C-JUN and CPP32 (CASPASE 3) in human pancreatic cancer: relation to cell proliferation and death[J]. Pancreas, 2003, 26(1): 65-70.
2. Roy S, Bayly CI, Gareau Y, et al. Maintenance of caspase-3 proenzyme dormancy by an intrinsic “safety catch” regulatory tripeptide[J]. Proc Natl Acad Sci USA, 2001, 98(11): 6132-6137.
3. 刘玉峰, 贾国存, 曾利, 等. 铁剥夺诱导HL-60细胞凋亡及对化疗药物诱导HL-60细胞凋亡的影响[J]. 中华儿科杂志, 2001, 39(12): 735-738.
4. Yang R, Muller C, Huynh V, et al. Functions of cyclin A1 in the cell cycle and its interactions with transcription factor E2F-1 and the Rb family of proteins[J]. Mol Cell Biol, 1999, 19(3): 2400-2407.
5. Epsztejn S, Kakhlon O, Glickstein H, et al. Fluorescence analysis of the labile iron pool of mammalian cells[J]. Anal Biochem, 1997, 248(1): 31-40.
6. Palozza P, Serini S, Torsello A, et al. Mechanism of activation of caspase cascade during beta-carotene-induced apoptosis in human tumor cells[J]. Nutr Cancer, 2003, 47(1): 76-87.
7. Keri G, Racz G, Magyar K, et al. Pro-apoptotic and anti-apoptotic molecules affecting pathways of signal transduction[J]. Ann NY Acad Sci, 2003, 1010(12): 109-112.
8. Roy S, Bayly CI, Gareau Y, et al. Maintenance of caspase-3 proenzyme dormancy by an intrinsic “safety catch” regulatory tripeptide[J]. Proc Natl Acad Sci USA, 2001, 98(11): 6132-6137.
9. 黄贵清, 廖清奎, 罗春华. 急性白血病患儿外周血白血病细胞转铁蛋白受体表达分析[J]. 华西医科大学学报, 1997, 28(1): 55-57.
10. 贾国存, 汤有才, 李丰益, 等. HL-60细胞铁池变化与凋亡相关基因关系的初步研究[J]. 中国小儿血液与肿瘤杂志, 2007, 5(12): 209-212.
11. 刘玉峰, 王叨, 张传新. 去铁胺诱导白血病细胞HL-60凋亡线粒体膜电位变化的研究[J]. 中华儿科杂志, 2005, 43(4): 300-301.
12. Fang D, Bao Y, Li X, Liu F. Effects of iron deprivation on multidrug resistance of leukemic K562 cells[J]. Chemotherapy, 2010, 56(1): 9-16.
13. Noulsri E, Richardson DR, Lerdwana S. et al. Antitumor activity and mechanism of action of the iron chelator, Dp44mT, against leukemic cells[J]. Am J Hematol, 2009, 84(3): 170-176.
14. Mori S, Sawada T, Okada T, et al. Anti-proliferative effect of interferon-gamma is enhanced by iron chelation in colon cancer cell lines in vitro[J]. Hepatogastroenterology, 2008, 55(85): 1274-1279.
15. Kontoghiorghes GJ, Efstathiou A, Ioannou-Loucaides S, et al. Chelators controlling metal metabolism and toxicity pathways: applications in cancer prevention, diagnosis and treatment[J]. Hemoglobin, 2008, 32(1-2): 217-227.

West China Medical Journal

Changes of Caspase-3 in Deferoxamine-Induced Apoptosis of HL-60 Cells

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