[Mechanism of sodium nitroprusside-induced apoptosis in K562 cell line].
To study the molecular mechanisms of nitric oxide donor sodium nitroprusside (SNP) -induced apoptosis in K562 human leukemia cell line, the different concentrations of SNP and different time of culture were used to treat K562 cell. At the same time, potassium ferricyamide (PFC) was used as control, blank was designed in experiment. Cell apoptosis was analysed by cell morphology, DNA agarose gel electrophoresis, DNA content, and annexin-V/PI labeling method. The TdT-mediated dUTP nick end labeling (TUNEL) assay was used to quantify in situ cell apoptosis. Reactive oxygen species (ROS) in cells and mitochondrial transmembrane potential (DeltaPsim) were labeled by dihydrorhodamin 123, 2', 7'-dichlorodihydrofluorescein diacetate and rhodamin 123/PI. bcl-2, bax, bad, p53 gene proteins and mitochondrial membrane protein were analysed by flow cytometry. The results showed that the K562 cell apoptosis was confirmed by typical cell morphology, DNA fragment, sub-G(1) phase, TUNEL and annexin-V/PI labeling. A majority of K562 cells were arrested in G(0)/G(1) phase. During the process of SNP-induced apoptosis in K562 cell, the mean fluorescence intensity of ROS in cells was significantly higher than those in blank and PFC control, while the DeltaPsim reduced. The expression of p53, bax, bad, Fas protein and mitochondrial membrane protein increased and bcl-2 protein decreased after SNP treatment. It is concluded that SNP induces K562 cell apoptosis through increasing ROS in cells, expressing the p53, bax, bad, Fas protein and mitochondrial membrane protein and decreasing bcl-2 protein, opening the mitochondrial permeability transition pore and reducing DeltaPsim. Furthermore, the Fas was activated during the apoptosis process.
Zhou
Yong-Lie
YL
Central Laboratory, Zhejiang Provincial People's Hospital, Hangzhou 310014, China. zyl@zjyxjy.com
Lü
Ya-Ping
YP
Qiu
Lian-Nü
LN
Wang
Wen-Song
WS
Lin
Hui-Jun
HJ
D017209:Apoptosis / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D020287:In Situ Nick-End Labeling; D020014:K562 Cells; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D009599:Nitroprusside / Q000494:pharmacology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism; D019014:fas Receptor / Q000096:biosynthesis
D020030:Nitric Oxide Donors; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D019014:fas Receptor; D009599:Nitroprusside