Using FRET Technique to Investigate The Apoptotic Mechanism Induced by Cisplatin in Living Cells

作者：Liu Lei^*; Zhang Ying Jie; Wang Xian Wang

来源：Progress in Biochemistry and Biophysics, 2009, 36(9): 1172-1179.

DOI：10.3724/SP.J.1206.2009.00067

• FRET
• cisplatin
• apoptosis
• Bid
• Caspase-3

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 similar to 5 h after treated with 20 mu 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.

• 出版日期2009-9
• 单位华南师范大学