Journal Article

Activation of caspase-3 activity and apoptosis in MDA-MB-468 cells by <i>N</i><sup>ω</sup>-hydroxy-L-arginine, an inhibitor of arginase, is not solely dependent on reduction in intracellular polyamines

Rajan Singh, Shehla Pervin, Guoyao Wu and Gautam Chaudhuri

in Carcinogenesis

Volume 22, issue 11, pages 1863-1869
Published in print November 2001 | ISSN: 0143-3334
Published online November 2001 | e-ISSN: 1460-2180 | DOI: http://dx.doi.org/10.1093/carcin/22.11.1863
Activation of caspase-3 activity and apoptosis in MDA-MB-468 cells by Nω-hydroxy-L-arginine, an inhibitor of arginase, is not solely dependent on reduction in intracellular polyamines

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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 α-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 G0–G1 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.

Keywords: DFMO, α-difluoromethylornithine; NOHA, Nω-hydroxy-l-arginine; NO, nitric oxide; NOS, nitric oxide synthase; ODC, ornithine decarboxylase; SAMDC, S-adenosylmethionine decarboxylase.

Journal Article.  5544 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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