title: Activation of caspase-3 activity and apoptosis in MDA-MB-468 cells by N(omega)-hydroxy-L-arginine, an inhibitor of arginase, is not solely dependent on reduction in intracellular polyamines.
abstract: We have shown previously that (NOHA) an intermediate in the nitric oxide (NO) synthetic pathway and an inhibitor of arginase significantly reduced intracellular polyamines, activated caspase-3 and induced apoptosis in the human breast cancer cell line MDA-MB-468. These actions of NOHA were abolished in the presence of exogenous L-ornithine suggesting that a reduction in the intracellular polyamine content might be responsible for the activation of caspase-3 and apoptotic actions of NOHA. In order to further explore this possibility, we used SAM-486A and alpha-difluoromethylornithine (DFMO), which are inhibitors of S-adenosylmethionine decarboxylase (SAMDC), and ornithine decarboxylase (ODC), respectively, either alone or in combination to reduce the intracellular polyamine levels. We then assessed whether a reduction in polyamine levels by these two compounds to a similar degree to that produced by NOHA activated caspase-3 which occurs prior to the onset of apoptosis. We observed that both SAM-486A and DFMO, either alone or in combination, inhibited cell proliferation, induced p21 and arrested cells in the G(0)-G(1) phase of the cell cycle but failed to activate caspase-3 as assessed by enzymatic assay of caspase-3, western blot analysis of the proteolytic cleavage of caspase-3 protein as well as TUNEL assay. Furthermore, pre-incubation of the cells with SAM-486A and DFMO for 4 days, either alone or in combination significantly inhibited the activation of caspase-3 and apoptosis by NOHA when compared with that observed with cells treated with NOHA alone. Our results, therefore, indicate that the activation of caspase-3 and apoptosis observed with NOHA cannot be solely explained by a reduction in intracellular polyamine levels and that other mechanisms need to be also considered.
authors.affiliation: Department of Obstetrics, Jonsson Comprehensive Cancer Center, University of California School of Medicine, Los Angeles, CA 90095-1740, USA.
authors.forename: R
authors.initials: R
authors.lastname: Singh
authors.forename: S
authors.initials: S
authors.lastname: Pervin
authors.forename: G
authors.initials: G
authors.lastname: Wu
authors.forename: G
authors.initials: G
authors.lastname: Chaudhuri
mesh_terms: D012437:Adenosylmethionine Decarboxylase / Q000037:antagonists & inhibitors; D017209:Apoptosis; D001119:Arginase / Q000037:antagonists & inhibitors*; D001120:Arginine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D020287:In Situ Nick-End Labeling; D009952:Ornithine / Q000494:pharmacology; D009955:Ornithine Decarboxylase / Q000378:metabolism; D011073:Polyamines / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology
chemical_list: C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004791:Enzyme Inhibitors; D011073:Polyamines; C068309:N(omega)-hydroxyarginine; D001120:Arginine; D009952:Ornithine; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D001119:Arginase; D009955:Ornithine Decarboxylase; D012437:Adenosylmethionine Decarboxylase