Using FRET Technique to Investigate The Apoptotic Mechanism Induced by Cisplatin in Living Cells
- VernacularTitle:利用荧光共振能量转移技术在活细胞内研究顺铂诱导的凋亡信号通路
- Author:
Lei LIU
;
Yingjie ZHANG
;
Xianwang WANG
- Publication Type:Journal Article
- Keywords:
FRET, cisplatin, apoptosis, Bid, Caspase-3
- From:
Progress in Biochemistry and Biophysics
2006;0(09):-
- CountryChina
- Language:Chinese
-
Abstract:
Cisplatin, an efficient anticancer agent, can trigger multiple apoptotic pathways in cancer cell. However, the signal transduction pathways in response to cisplatin-based chemotherapy are complicated, and the mechanism is not fully understood. Using fluorescence resonance energy transfer (FRET) technique, the molecular mechanism of cisplatin-induced apoptosis in living human lung adenocarcinoma cells (ASTC-a-1) were investigated. After cisplatin treatment, the recombinant pFRET-Bid and pSCAT-3 probes were used to determine the kinetics of Bid cleavage and Caspase-3 activation, respectively. The fluorescence probes Bid-CFP and DsRed-Mit were also used to detect the spatial and temporal changes of Bid in real-time in sub-cell level. The results showed that a cleavage of the Bid-FRET probe occurring at about 4～5 h after treated with 20 ?mol/L cisplatin. Cleavage of the Bid-FRET probe coincided with a translocation of tBid from the cytosolic to the mitochondria, and the translocation lasted about 1.5 h. At the anaphase of cell apoptosis, Caspase-3 was activated obviously as detected by FRET and Western blotting techniques. Using real-time single-cell analysis, it was observed the kinetics of Bid and Caspase-3 activation for the first time in living cells during cisplatin-induced apoptosis.
