cytochrome-c-t and methoxyamine

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.. 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 blot.. The dose-response curve of manumycin was shifted to the left after addition of methoxyamine. The combination index (CI) was less than 1 (P<0.05) in U937 cells (P<0.05), indicating a synergistic effect of manumycin and methoxyamine. Rates of colony formation of U937 cells treated with 1 micromol/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 (P<0.05). 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.. Methoxyamine enhances manumycin-induced apoptosis in U937 myeloid leukemia cells.

Topics: Antineoplastic Agents; Apoptosis; Caspase 9; Cytochromes c; Drug Synergism; Enzyme Inhibitors; Farnesyltranstransferase; Humans; Hydroxylamines; Mitochondria; Poly(ADP-ribose) Polymerases; Polyenes; Polyunsaturated Alkamides; U937 Cells

Farnesyltransferase inhibitors (FTIs) are currently under investigation for leukemia treatment. We evaluated the FTI manumycin A (manumycin) in two myeloid leukemia cell lines (U937 and HL-60). Manumycin induced nitric oxide production and apoptosis of the leukemia cells. Nitric oxide or other reactive oxygen species may induce oxidative DNA damage, and the number of apurinic sites increased after manumycin treatment, which was reversed by concurrent treatment with N-acetyl-L-cysteine. Since repair of DNA damage is important to cell survival, we hypothesized that methoxyamine, an inhibitor of base-excision repair, would enhance the antineoplastic effect of manumycin. The combination of manumycin and methoxyamine resulted in enhanced apoptosis by six criteria increased annexin V binding, release of mitochondrial cytochrome c into the cytosol, activation of caspase-9, activation of caspase-3, specific cleavage of poly-adenosyl ribose polymerase, and increase in the sub-G1 cell cycle fraction. The drug combination enhanced inhibition on the soft agar clonogenic assay and on the formazan dye cell viability assay. The effects of manumycin or manumycin plus methoxyamine on apoptosis were blocked by N-acetyl-L-cysteine, and partially by nitric oxide synthase inhibitors or scavenger of peroxide. We conclude that methoxyamine enhances manumycin-induced apoptosis in myeloid leukemia cells.

Topics: Apoptosis; Caspase 3; Caspase 9; Caspases; Cell Survival; Cytochromes c; Cytosol; DNA, Neoplasm; Drug Synergism; Enzyme Inhibitors; HL-60 Cells; Humans; Hydroxylamines; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Mitochondria; Nitric Oxide; Poly(ADP-ribose) Polymerases; Polyenes; Polyunsaturated Alkamides; U937 Cells