Objective: To investigate the apoptosis induced by manumycin in U937 and HL-60 cell lines, and to explore the role of mitochondria apoptotic pathway in manumycin-inducing apoptosis.
Methods: Leukemic cells line U937 and HL-60 were treated by manumycin at 2 micromol/L for different time. Apoptosis of leukemia cells was detected by flow cytometry. The cytosolic proteins were extracted using a digitonin buffer. The protein expression of cytochrome C, caspase-9, caspase-8, and caspase-3 were determined by western blot. Mitochondrial membrane potential was detected by JC-1.
Results: In U937 and HL-60 cells, manumycin induced mitochondrial depolarization after 6 h treatment. The average red/green fluorescence ratios at 6 h were significantly (P < 0.01) lower than those at time 0, being 0.51 +/- 0.07 and 0.41 +/- 0.06 for control group respectively. Manumycin induced cytochrome C release from the mitochondria into the cytosol after 6 h treatment, and activated caspase-9, caspase-8, and caspase-3 after a 16h treatment. The broad-spectrum caspase-inhibitor Z-VAD-fmk at 50 micromol/L was able to inhibit caspase cleavage completely, but only reduced the manumycin-induced apoptosis rates by 51.69% and 56.47% in U937 and HL-60, respectively.
Conclusion: Manumycin induced apoptosis in U937 and HL-60 cell lines via mitochondria apoptotic pathway.