Glutathione oxidation and mitochondrial depolarization as mechanisms of nordihydroguaiaretic acid-induced apoptosis in lipoxygenase-deficient FL5.12 cells
Ss. Biswal et al., Glutathione oxidation and mitochondrial depolarization as mechanisms of nordihydroguaiaretic acid-induced apoptosis in lipoxygenase-deficient FL5.12 cells, TOXICOL SCI, 53(1), 2000, pp. 77-83
Nordihydroguaiaretic acid (NDGA) induces apoptosis in a variety of cell lin
es. The mechanism(s) of this effect is not known, although the focus has be
en on the ability of NDGA to inhibit lipoxygenase (LOX) activities. In the
present study, NDGA-induced apoptosis was studied in a murine hematopoietic
cell line, FL5.12. Although this cell line lacks detectable LOX protein or
activities, NDGA(10 mu M) was able to induce apoptosis, There was a massiv
e loss of mitochondrial membrane potential by 4 h after the addition of NDG
A, suggesting that this organelle might be targeted by NDGA. A pro-oxidant
NDGA effect has been suggested as playing a role in apoptosis. This was sup
ported by the findings that glutathione disulfide levels were increased by
4 h following treatment with 10 mu M NDGA, that pretreatment with N-acetylc
ysteine completely blocked the NDGA-induced loss of membrane potential and
apoptosis, and that lipid peroxidation was enhanced in cells treated with N
DGA. However, no evidence of increased levels of reactive oxygen could be s
een in NDGA-treated cells loaded with dichlorofluorescin diacetate or dihyd
rorhodamine and analyzed by flow cytometry. Bcl-x(L) protein levels were un
affected by NDGA treatment. Caspase-3 was rapidly activated with a peak at
8 h after FL5.12 cells were treated with NDGA, Ac-DEVD-CHO (25 mu M) and bo
c-asp-FMK (20 mu M) both inhibited caspase-3 enzyme activity by 97% 8 h aft
er NDGA treatment. Boc-asp-FMK, a more general caspase inhibitor, delayed N
DGA-induced apoptosis while Ac-DEVD-CHO, a more specific inhibitor of caspa
se-3, had no effect. These results suggest that NDGA-induced apoptosis happ
ens through reactions that depolarize mitochondria, oxidize glutathione and
lipids, but do not generate significant amounts of free reactive oxygen sp
ecies.