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.
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.
Singh
R
R
Department of Obstetrics, Jonsson Comprehensive Cancer Center, University of California School of Medicine, Los Angeles, CA 90095-1740, USA.
Pervin
S
S
Wu
G
G
Chaudhuri
G
G
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
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