Generation of a Stable Antioxidant Response Element–Driven Reporter Gene Cell Line and Its Use to Show Redox-Dependent Activation of Nrf2 by Cancer Chemotherapeutic Agents

Abstract

The NF-E2 p45-related factor 2 (Nrf2) regulates cytoprotective genes that contain an antioxidant response element (ARE) in their promoters. To investigate whether anticancer drugs can induce ARE-driven gene expression, we have developed a stable human mammary MCF7-derived reporter cell line called AREc32, which contains a luciferase gene construct controlled by eight copies of the cis-element. In these cells, luciferase activity was increased up to 50-fold following treatment with 50 Mmol/L tert-butylhydroquinone (t-BHQ). Basal and inducible luciferase activities in AREc32 cells were increased by forced overexpression of Nrf2 and reduced by knockdown of endogenous Nrf2 expression with RNA interference. Depletion of cellular reduced glutathione (GSH) by treatment of AREc32 cells with L-buthionine-S,R -sulfoximine (BSO) did not influence basal levels of luciferase activity, but pretreatment with BSO augmented induction of luciferase activity by t-BHQ. Induction of reporter activity by t-BHQ in AREc32 cells was suppressed markedly by the antioxidants N-acetylcysteine and GSH but only modestly by vitamins C or E, suggesting that ARE-luciferase expression is induced primarily by thiol-active electrophiles rather than free radicals. The anticancer drugs cisplatin, etoposide, mitoxantrone, chlorambucil, melphalan, and carmustine [1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU)] weakly induced luciferase activity in AREc32 cells. Moreover, treatment of AREc32 cells with BSO immediately before exposure to anticancer drugs enhanced induction of ARE-driven luciferase activity by cisplatin, BCNU, chlorambucil, and melphalan and also induced endogenous AKR1C (AKR1C refers to AKR1C1 and AKR1C2), a target gene of Nrf2. Our findings show that Nrf2 can be activated by certain anticancer agents, and this will influence the effectiveness of chemotherapy. (Cancer Res 2006; 66(22): 10983-94)

Electrophilic tuning of the chemoprotective natural product sulforaphane

“…S4B). As expected, cellular depletion of glutathione with L-buthionine-sulfoximine enhanced luciferase induction by 8f and 4o in a fashion comparable to that of sulforaphane (33) (Fig. S4C).…”

“…To confirm that, like sulforaphane, the sulfoxythiocarbamates stimulate NQO1 via the antioxidant response element (ARE) transcriptional pathway, we examined eight sulfoxythiocarbamate analogs by using a previously established ARE-luciferase reporter stably transfected in MCF7 cells (AREc32) (33). Each of these compounds led to a significant increase in the luciferase signal, and the most powerful effects were caused by 8f and 4o as shown in Fig.…”

Oxidative and electrophilic stress induces multidrug resistance-associated protein transporters via the nuclear factor-E2-related factor-2 transcriptional pathway

“…22,39 Although GSH depletion and Nrf2 activation are known to be linked, the exact mechanism of how this process occurs remains unclear at present. A recent report by Wang et al 40 demonstrated that the depletion of GSH with BSO was not sufficient to drive the expression of an ARE promoter/ reporter construct, yet BSO combined with other Nrf2 activators led to the synergistic activation of this construct. This may suggest that although GSH depletion does not cause Nrf2 activation directly, the attenuation of the GSH buffering system may make the Nrf2/Keap1 pathway more susceptible to chemical activation by strong electrophiles.…”

NRF2 inhibition causes repression of ATM and ATR expression leading to aberrant DNA Damage Response

“…To determine NRF2 dependent transcriptional antioxidant response following different treatments, stable clones of MCF7 cells carrying PGL3 vector with a cloned 8 copies of Cis-Antioxidant Response Elements (ARE) reporter construct (AREc32) was used [45]. Briefly, AREc32 was seeded in 96 well plates at a density of 1.5×10 4 cells per well and allowed to attach for 18h.…”