Effects of manumycin combined with methoxyamine on apoptosis in myeloid leukemia U937 cells

BACKGROUND OBJECTIVE:Repair of DNA damage is important to cell survival.Our previous study showed DNA damage response induced by manumycin in cancer cells.We hypothesized that methoxyamine,an inhibitor of base-excision repair,can enhance the antineoplastic effect of manumycin.This study was to investigate apoptosis induced by manumycin combined with methoxyamine in myeloid leukemia cell line U937,and to explore the role of mitochondrial apoptotic pathway in apoptosis induction of the two drugs.METHODS:U937 cells were treated with various concentrations of manumycin and/or methoxyamine for 48 h.The cell viability was analyzed by MTT assay.Colony formation was evaluated by soft agar clonogenic assay.Cell apoptosis was investigated by flow cytometry.Protein expressions of cytochrome c,caspase-9,and poly ADP-ribose polymerase(PARP) were determined by Western blott.RESULTS:The dose-response curve of manumycin was shifted to the left after addition of methoxyamine.The combination index(CI) was less than 1(P0.05) in U937 cells(P0.05),indicating a synergistic effect of manumycin and methoxyamine.Rates of colony formation of U937 cells treated with 1μmol/L manumycin,or 5 mmol/L methoxyamine,or the combination of the two were 0.3641±0.0463,0.7541±0.0379,and 0.0473±0.0024,respectively compared with that of control cells(P0.05).Moreover,the drug combination resulted in enhanced apoptosis in U937 cells.The apoptotic rates of the control,manumycin,methoxyamine and combination group were(2.34±0.30)%,(8.80±0.95)%,(2.21±0.19)%,and(13.37±0.91)%,respectively.The combination of manumycin with methoxyamine also promoted the release of cytochrome c from mitochondria into the cytosol,activated caspase-9,and led appearance of specific cleavage of PARP in U937 cells.CONCLUSION:Methoxyamine enhances manumycin-induced apoptosis in U937 myeloid leukemia cells.