The Antioxidant Transcription Factor Nrf2 Negatively Regulates Autophagy and Growth Arrest Induced by the Anticancer Redox Agent Mitoquinone

V Ashutosh Rao,Sarah R Klein,Spencer J Bonar,Jacek Zielonka,Naoko Mizuno,Jennifer S Dickey,Paul W Keller,Joy Joseph,Balaraman Kalyanaraman,Emily Shacter

doi:10.1074/jbc.m110.133579

Abstract

Mitoquinone (MitoQ) is a synthetically modified, redox-active ubiquinone compound that accumulates predominantly in mitochondria. We found that MitoQ is 30-fold more cytotoxic to breast cancer cells than to healthy mammary cells. MitoQ treatment led to irreversible inhibition of clonogenic growth of breast cancer cells through a combination of autophagy and apoptotic cell death mechanisms. Relatively limited cytotoxicity was seen with the parent ubiquinone coenzyme Q(10.) Inhibition of cancer cell growth by MitoQ was associated with G(1)/S cell cycle arrest and phosphorylation of the checkpoint kinases Chk1 and Chk2. The possible role of oxidative stress in MitoQ activity was investigated by measuring the products of hydroethidine oxidation. Increases in ethidium and dihydroethidium levels, markers of one-electron oxidation of hydroethidine, were observed at cytotoxic concentrations of MitoQ. Keap1, an oxidative stress sensor protein that regulates the antioxidant transcription factor Nrf2, underwent oxidation, degradation, and dissociation from Nrf2 in MitoQ-treated cells. Nrf2 protein levels, nuclear localization, and transcriptional activity also increased following MitoQ treatment. Knockdown of Nrf2 caused a 2-fold increase in autophagy and an increase in G(1) cell cycle arrest in response to MitoQ but had no apparent effect on apoptosis. The Nrf2-regulated enzyme NQO1 is partly responsible for controlling the level of autophagy. Keap1 and Nrf2 act as redox sensors for oxidative perturbations that lead to autophagy. MitoQ and similar compounds should be further evaluated for novel anticancer activity.

Highlights

Mitoquinone (MitoQ) is a synthetically modified, redox-active ubiquinone compound that accumulates predominantly in mitochondria
We found that mitoquinone has significant and selective anticancer activity, unlike untargeted ubiquinone (CoQ10)
The NQO1-negative cells showed a greater percentage of cells with autophagosomes as seen by microscopy (Fig. 7F). These results suggest that NQO1 is, at least in part, responsible for the downstream signaling from Nrf2 and controlling the level of autophagy in response to MitoQ

Summary

The abbreviations used are

MitoQ, mitoquinone; ARE, antioxidant response element; CoQ10, coenzyme Q-10 or ubiquinone; HE, hydroethidine; ROS, reactive oxygen species; tBHQ, tert-butylhydroquinone; VP-16, etoposide; PI, propidium iodide; SRB, sulforhodamine B; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; JC-1, 5,5Ј,6,6Ј-tetrachloro1,1Ј,3,3Ј-tetraethylbenzimidazolcarbocyanine iodide. The role of mitochondrial oxidative phosphorylation and signaling in cancer cells is complex and controversial [11, 12]. Cancer cells have a greater mitochondrial membrane potential than healthy cells [13]. Rhodamine 123, a lipophilic cationic probe, selectively accumulates in the mitochondria and induces greater toxicity in cancer cells [14, 15]. We found that mitoquinone has significant and selective anticancer activity, unlike untargeted ubiquinone (CoQ10). We demonstrate that the Nrf2-Keap proteins act as redox sensors that relay the signal for a failed cytoprotective response to MitoQ. Data are provided to elucidate the cellular response to MitoQ and to determine its novel underlying mechanism of action

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION