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Involvement of mitochondria apoptotic pathway in the manumycin inducing apoptosis of U937 and HL-60

SHE Miao-rong LI Jin-gao DU Xin LIN Wei NIU Xin-qing GUO Kun-yuan Department of Hematology,Guangdong Provincial People's Hospital,Guangzhou 510080,China  
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 μmol/L for different time.Apoptosis of leukemia cells was detected by flow cytometry.The cytosolic proteins were extracted using a digitonin buff- er.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,manumy- cin induced mitochondrial depolarization after 6 h treatment.The average red/green fluorescence ratios at 6 h were significantly(P0.01)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 μmol/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.Conclu- sion Manumycin induced apoptosis in U937 and HL-60 cell lines via mitochondria apoptotic pathway.
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