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Cited by 233Genomics, phytochemicals, and oxidative stressBioactive Compounds and Antitumor AgentsDrug-Induced Hepatotoxicity and Protection

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Brusatol provokes a rapid and transient inhibition of Nrf2 signaling and sensitizes mammalian cells to chemical toxicity—implications for therapeutic targeting of Nrf2

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Olayanju, Adedamola, Copple, Ian M., Bryan, Holly K., Edge, George T., Sison, Rowena L., Wong, Min Wei, Lai, Zheng-Quan, Lin, Zhi-Xiu, Dunn, Karen, Sanderson, Christopher M., Alghanem, Ahmad F., Cross, Michael J., Ellis, Ewa C., Ingelman-Sundberg, Magnus, Malik, Hassan Z., Kitteringham, Neil R., Goldring, Christopher E., Park, B. Kevin·2015·Free Radical Biology and Medicine
DOI 10.1016/j.freeradbiomed.2014.11.003PMC PMC4291150
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Abstract

The transcription factor Nrf2 regulates the basal and inducible expression of a battery of cytoprotective genes. Whereas numerous Nrf2-inducing small molecules have been reported, very few chemical inhibitors of Nrf2 have been identified to date. The quassinoid brusatol has recently been shown to inhibit Nrf2 and ameliorate chemoresistance in vitro and in vivo. Here, we show that brusatol provokes a rapid and transient depletion of Nrf2 protein, through a posttranscriptional mechanism, in mouse Hepa-1c1c7 hepatoma cells. Importantly, brusatol also inhibits Nrf2 in freshly isolated primary human hepatocytes. In keeping with its ability to inhibit Nrf2 signaling, brusatol sensitizes Hepa-1c1c7 cells to chemical stress provoked by 2,4-dinitrochlorobenzene, iodoacetamide, and N-acetyl-p-benzoquinone imine, the hepatotoxic metabolite of acetaminophen. The inhibitory effect of brusatol toward Nrf2 is shown to be independent of its repressor Keap1, the proteasomal and autophagic protein degradation systems, and protein kinase signaling pathways that are known to modulate Nrf2 activity, implying the involvement of a novel means of Nrf2 regulation. These findings substantiate brusatol as a useful experimental tool for the inhibition of Nrf2 signaling and highlight the potential for therapeutic inhibition of Nrf2 to alter the risk of adverse events by reducing the capacity of nontarget cells to buffer against chemical and oxidative insults. These data will inform a rational assessment of the risk:benefit ratio of inhibiting Nrf2 in relevant therapeutic contexts, which is essential if compounds such as brusatol are to be developed into efficacious and safe drugs.

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Fig. 1 Rapid and transient posttranscriptional inhibition of Nrf2 by brusatol. (A and B) Nrf2 protein levels in whole-cell lysates prepared from Hepa-1c1c7 cells exposed to (A) the indicated concentrations of brusatol for 2 h or (B) 300 nM brusatol for the indicated times, as determined by immunoblotting. All blots shown are representative of three independent experiments. Nrf2 levels were quantified by densitometry, normalized to the loading control, β-actin, and expressed as a percentage of the Nrf2 level detected (A) in vehicle-exposed cells or (B) at 0 h. (C) Nrf2 mRNA level in Hepa-1c1c7 cells exposed to 300 nM brusatol for the indicated times, as determined by RT-qPCR. Nrf2 levels were normalized to the loading control, Gapdh, and expressed as a percentage of the Nrf2 mRNA level detected at 0 h. (D) Superimposition of the time-course data for Nrf2 protein and mRNA presented in (B) and (C). (E) ATP content in Hepa-1c1c7 cells exposed to the indicated concentrations of brusatol for 2 or 24 h. ATP levels are expressed as a percentage of the ATP content of vehicle-exposed cells. All data represent the mean + SD of n=3 independent experiments. Statistical analyses were performed using an unpaired t test (*P<0.05, **P<0.01, ***P<0.001 vs vehicle or 0 h control).
Fig. 1 Rapid and transient posttranscriptional inhibition of Nrf2 by brusatol. (A and B) Nrf2 protein levels in whole-cell lysates prepared from Hepa-1c1c7 cells exposed to (A) the indicated concentrations of brusatol for 2 h or (B) 300 nM brusatol for the indicated times, as determined by immunoblotting. All blots shown are representative of three independent experiments. Nrf2 levels were quantified by densitometry, normalized to the loading control, β-actin, and expressed as a percentage of the Nrf2 level detected (A) in vehicle-exposed cells or (B) at 0 h. (C) Nrf2 mRNA level in Hepa-1c1c7 cells exposed to 300 nM brusatol for the indicated times, as determined by RT-qPCR. Nrf2 levels were normalized to the loading control, Gapdh, and expressed as a percentage of the Nrf2 mRNA level detected at 0 h. (D) Superimposition of the time-course data for Nrf2 protein and mRNA presented in (B) and (C). (E) ATP content in Hepa-1c1c7 cells exposed to the indicated concentrations of brusatol for 2 or 24 h. ATP levels are expressed as a percentage of the ATP content of vehicle-exposed cells. All data represent the mean + SD of n=3 independent experiments. Statistical analyses were performed using an unpaired t test (*P<0.05, **P<0.01, ***P<0.001 vs vehicle or 0 h control).CC BY · PubMed Central
Fig. 2 Functional consequences of Nrf2 inhibition by brusatol. The mRNA levels of the Nrf2 target genes Nqo1 and Gclm in Hepa-1c1c7 cells exposed to 300 nM brusatol for the indicated times are shown, as determined by RT-qPCR. Nqo1 and Gclm mRNA levels were normalized to the loading control, Gapdh, and expressed as a percentage of the Nqo1 and Gclm levels detected at 0 h. All data represent the mean + SD of n=3 independent experiments. Statistical analyses were performed using an unpaired t test (*P<0.05, **P<0.01, ***P<0.001 vs 0-h control).
Fig. 2 Functional consequences of Nrf2 inhibition by brusatol. The mRNA levels of the Nrf2 target genes Nqo1 and Gclm in Hepa-1c1c7 cells exposed to 300 nM brusatol for the indicated times are shown, as determined by RT-qPCR. Nqo1 and Gclm mRNA levels were normalized to the loading control, Gapdh, and expressed as a percentage of the Nqo1 and Gclm levels detected at 0 h. All data represent the mean + SD of n=3 independent experiments. Statistical analyses were performed using an unpaired t test (*P<0.05, **P<0.01, ***P<0.001 vs 0-h control).CC BY · PubMed Central
Fig. 3 Inhibition of chemically induced Nrf2 by brusatol. (A and B) Nrf2 protein levels in whole-cell lysates prepared from Hepa-1c1c7 cells exposed to (A) vehicle or 300 nM brusatol for 2 h, followed by the indicated concentrations of CDDO-Me for 1 h, or (B) the indicated concentrations of CDDO-Me for 1 h, followed by 300 nM brusatol for 2 h, as determined by immunoblotting. (C) Nrf2–Venus levels in SK-N-AS cells exposed to 100 nM CDDO-Me for 1 h, followed by 300 nM brusatol for the indicated times. Still images were captured from the real-time data presented in Supplementary Video 1. (D and E) Nrf2 protein levels in whole-cell lysates prepared from Hepa-1c1c7 cells exposed to vehicle or 300 nM brusatol for 2 h, followed by the indicated concentrations of (D) NEM or (E) IAA for 1 h. All blots shown are representative of three independent experiments. Nrf2 levels were quantified by densitometry, normalized to the loading control, β-actin, and expressed as a percentage of the Nrf2 level detected in vehicle-exposed cells. Data represent the mean + SD of n=3 independent experiments. Statistical analysis was performed using an unpaired t test (*P<0.05, **P<0.01 vs CDDO-Me, IAA, or NEM alone).
Fig. 3 Inhibition of chemically induced Nrf2 by brusatol. (A and B) Nrf2 protein levels in whole-cell lysates prepared from Hepa-1c1c7 cells exposed to (A) vehicle or 300 nM brusatol for 2 h, followed by the indicated concentrations of CDDO-Me for 1 h, or (B) the indicated concentrations of CDDO-Me for 1 h, followed by 300 nM brusatol for 2 h, as determined by immunoblotting. (C) Nrf2–Venus levels in SK-N-AS cells exposed to 100 nM CDDO-Me for 1 h, followed by 300 nM brusatol for the indicated times. Still images were captured from the real-time data presented in Supplementary Video 1. (D and E) Nrf2 protein levels in whole-cell lysates prepared from Hepa-1c1c7 cells exposed to vehicle or 300 nM brusatol for 2 h, followed by the indicated concentrations of (D) NEM or (E) IAA for 1 h. All blots shown are representative of three independent experiments. Nrf2 levels were quantified by densitometry, normalized to the loading control, β-actin, and expressed as a percentage of the Nrf2 level detected in vehicle-exposed cells. Data represent the mean + SD of n=3 independent experiments. Statistical analysis was performed using an unpaired t test (*P<0.05, **P<0.01 vs CDDO-Me, IAA, or NEM alone).CC BY · PubMed Central
Fig. 4 Inhibition of Nrf2 by brusatol in primary human hepatocytes. Nrf2 and Nqo1 protein levels in whole-cell lysates prepared from freshly isolated primary human hepatocytes exposed to 100 nM CDDO-Me for 1 h, followed by 100 nM brusatol for the remainder of the indicated times, are shown as determined by immunoblotting. The proteasome inhibitor MG132 was used as a positive control for Nrf2 accumulation. The blots shown are from samples derived from donor PHH1 and are representative of experiments performed using cells isolated from four donors (PHH1–4). Nrf2 levels in cells exposed to CDDO-Me and brusatol for a total of 25 h were quantified by densitometry, normalized to the loading control, β-actin, and expressed as a percentage of the Nrf2 level detected in vehicle-exposed cells. Statistical analysis was performed using an unpaired t test (*P<0.05 vs CDDO-Me alone).
Fig. 4 Inhibition of Nrf2 by brusatol in primary human hepatocytes. Nrf2 and Nqo1 protein levels in whole-cell lysates prepared from freshly isolated primary human hepatocytes exposed to 100 nM CDDO-Me for 1 h, followed by 100 nM brusatol for the remainder of the indicated times, are shown as determined by immunoblotting. The proteasome inhibitor MG132 was used as a positive control for Nrf2 accumulation. The blots shown are from samples derived from donor PHH1 and are representative of experiments performed using cells isolated from four donors (PHH1–4). Nrf2 levels in cells exposed to CDDO-Me and brusatol for a total of 25 h were quantified by densitometry, normalized to the loading control, β-actin, and expressed as a percentage of the Nrf2 level detected in vehicle-exposed cells. Statistical analysis was performed using an unpaired t test (*P<0.05 vs CDDO-Me alone).CC BY · PubMed Central
Fig. 5 Brusatol sensitizes mammalian cells to chemical stress. ATP content in Hepa-1c1c7 cells exposed to 300 nM brusatol for the indicated times, followed by the indicated concentrations of (A) DNCB for 6 h, (B) IAA for 6 h, or (C) NAPQI for 12 h is shown. ATP levels are expressed as a percentage of the ATP content of vehicle-exposed cells. Data represent the mean + SD of n=3 independent experiments. Statistical analysis was performed using an unpaired t test (*P<0.05, **P<0.01 vs vehicle control).
Fig. 5 Brusatol sensitizes mammalian cells to chemical stress. ATP content in Hepa-1c1c7 cells exposed to 300 nM brusatol for the indicated times, followed by the indicated concentrations of (A) DNCB for 6 h, (B) IAA for 6 h, or (C) NAPQI for 12 h is shown. ATP levels are expressed as a percentage of the ATP content of vehicle-exposed cells. Data represent the mean + SD of n=3 independent experiments. Statistical analysis was performed using an unpaired t test (*P<0.05, **P<0.01 vs vehicle control).CC BY · PubMed Central
Fig. 6 Brusatol does not provoke the general depletion of short-half-life proteins in Hepa-1c1c7 cells. Cyclin A, HIF-1α, p53, and survivin protein levels in whole-cell lysates prepared from Hepa-1c1c7 cells exposed to 300 nM brusatol for the indicated times are shown, as determined by immunoblotting. All blots shown are representative of three independent experiments.
Fig. 6 Brusatol does not provoke the general depletion of short-half-life proteins in Hepa-1c1c7 cells. Cyclin A, HIF-1α, p53, and survivin protein levels in whole-cell lysates prepared from Hepa-1c1c7 cells exposed to 300 nM brusatol for the indicated times are shown, as determined by immunoblotting. All blots shown are representative of three independent experiments.CC BY · PubMed Central
Fig. 7 Inhibition of Nrf2 by brusatol is not dependent on the canonical mechanisms of Nrf2 degradation. (A) Keap1 protein levels in whole-cell lysates prepared from Hepa-1c1c7 cells exposed to 300 nM brusatol for the indicated times, as determined by immunoblotting. (B) Keap1 and Nrf2 protein levels in whole-cell lysates prepared from Hepa-1c1c7 cells transfected with a scrambled, nontargeting control siRNA (si-CON) or a Keap1-targeting siRNA (si-KEAP1) for 48 h, followed by exposure to the indicated concentrations of brusatol for 2 h. (C) Effects of incubation with the indicated concentrations of brusatol or the proteasome inhibitor MG132, for the indicated times, on the chymotrypsin (Suc-LLVY-AMC)-, trypsin (Boc-LSTR-AMC)-, and caspase (Z-LLE-AMC)-like activities of human 20S proteasome. (D) Nrf2 protein levels in whole-cell lysates prepared from Hepa-1c1c7 cells exposed to the indicated concentrations of MG132 for 1 h, followed by 300 nM brusatol for 2 h. (E) p62 protein levels in whole-cell lysates prepared from Hepa-1c1c7 cells exposed to 300 nM brusatol for the indicated times. Lysates prepared from mock-transfected (-ve) and p62-transfected (+ve) HEK293T cells were loaded as controls. (F) Nrf2 and p62 protein levels in whole-cell lysates prepared from Hepa-1c1c7 cells exposed to 30 nM bafilomycin A1 or 10 mM ammonium chloride (NH4Cl) for 16 h, followed by the indicated concentrations of brusatol for 2 h. All blots shown are representative of three independent experiments. Nrf2 levels were quantified by densitometry, normalized to the loading control, β-actin, and expressed as a percentage of the Nrf2 level detected in vehicle-exposed cells. Data represent the mean + SD of n=3 independent experiments. Statistical analysis was performed using an unpaired t test (*P<0.05, **P<0.01, ***P<0.001 vs vehicle control or MG132 alone).
Fig. 7 Inhibition of Nrf2 by brusatol is not dependent on the canonical mechanisms of Nrf2 degradation. (A) Keap1 protein levels in whole-cell lysates prepared from Hepa-1c1c7 cells exposed to 300 nM brusatol for the indicated times, as determined by immunoblotting. (B) Keap1 and Nrf2 protein levels in whole-cell lysates prepared from Hepa-1c1c7 cells transfected with a scrambled, nontargeting control siRNA (si-CON) or a Keap1-targeting siRNA (si-KEAP1) for 48 h, followed by exposure to the indicated concentrations of brusatol for 2 h. (C) Effects of incubation with the indicated concentrations of brusatol or the proteasome inhibitor MG132, for the indicated times, on the chymotrypsin (Suc-LLVY-AMC)-, trypsin (Boc-LSTR-AMC)-, and caspase (Z-LLE-AMC)-like activities of human 20S proteasome. (D) Nrf2 protein levels in whole-cell lysates prepared from Hepa-1c1c7 cells exposed to the indicated concentrations of MG132 for 1 h, followed by 300 nM brusatol for 2 h. (E) p62 protein levels in whole-cell lysates prepared from Hepa-1c1c7 cells exposed to 300 nM brusatol for the indicated times. Lysates prepared from mock-transfected (-ve) and p62-transfected (+ve) HEK293T cells were loaded as controls. (F) Nrf2 and p62 protein levels in whole-cell lysates prepared from Hepa-1c1c7 cells exposed to 30 nM bafilomycin A1 or 10 mM ammonium chloride (NH4Cl) for 16 h, followed by the indicated concentrations of brusatol for 2 h. All blots shown are representative of three independent experiments. Nrf2 levels were quantified by densitometry, normalized to the loading control, β-actin, and expressed as a percentage of the Nrf2 level detected in vehicle-exposed cells. Data represent the mean + SD of n=3 independent experiments. Statistical analysis was performed using an unpaired t test (*P<0.05, **P<0.01, ***P<0.001 vs vehicle control or MG132 alone).CC BY · PubMed Central
Fig. 8 Inhibition of Nrf2 by brusatol is not dependent on the activation of p38 MAPK, AKT, ERK1/2, or JNK1/2 signaling. (A) Nrf2 and phospho-p38 MAPK, AKT, ERK1/2, and JNK1/2 levels in whole-cell lysates prepared from Hepa-1c1c7 cells exposed to 300 nM brusatol for the indicated times, as determined by immunoblotting. (B) Nrf2 protein levels in whole-cell lysates prepared from Hepa-1c1c7 cells exposed to the indicated concentrations of SB203580 (p38 MAPK), LY294002 (AKT), CT99021 (GSK3β), or SP600125 (JNK1/2) or 5 µM U0126 (ERK1/2) for 1 h, followed by 300 nM brusatol for 2 h. All blots shown are representative of three independent experiments. Nrf2 or protein kinase levels were quantified by densitometry, normalized to the loading control, β-actin, and expressed as a percentage of the Nrf2 or protein kinase level detected at 0 h or in vehicle-exposed cells. Data represent the mean + SD of n=3 independent experiments. Statistical analysis was performed using an unpaired t test (*P<0.05, **P<0.01, ***P<0.001 vs vehicle or 0 h control).
Fig. 8 Inhibition of Nrf2 by brusatol is not dependent on the activation of p38 MAPK, AKT, ERK1/2, or JNK1/2 signaling. (A) Nrf2 and phospho-p38 MAPK, AKT, ERK1/2, and JNK1/2 levels in whole-cell lysates prepared from Hepa-1c1c7 cells exposed to 300 nM brusatol for the indicated times, as determined by immunoblotting. (B) Nrf2 protein levels in whole-cell lysates prepared from Hepa-1c1c7 cells exposed to the indicated concentrations of SB203580 (p38 MAPK), LY294002 (AKT), CT99021 (GSK3β), or SP600125 (JNK1/2) or 5 µM U0126 (ERK1/2) for 1 h, followed by 300 nM brusatol for 2 h. All blots shown are representative of three independent experiments. Nrf2 or protein kinase levels were quantified by densitometry, normalized to the loading control, β-actin, and expressed as a percentage of the Nrf2 or protein kinase level detected at 0 h or in vehicle-exposed cells. Data represent the mean + SD of n=3 independent experiments. Statistical analysis was performed using an unpaired t test (*P<0.05, **P<0.01, ***P<0.001 vs vehicle or 0 h control).CC BY · PubMed Central

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Authors
Olayanju, A., Copple, I. M., Bryan, H. K., Edge, G. T., Sison, R. L., Wong, M. W., Lai, Z.-Q., Lin, Z.-X., Dunn, K., Sanderson, C. M., Alghanem, A. F., Cross, M. J., Ellis, E. C., Ingelman-Sundberg, M., Malik, H. Z., Kitteringham, N. R., Goldring, C. E., & Park, B. K.
Affiliations
  • University of Liverpool (GB)
  • Chinese University of Hong Kong (HK)
  • Karolinska University Hospital (SE)
  • Aintree University Hospitals NHS Foundation Trust (GB)
Corresponding author
Goldring, Christopher E.
ORCID
Venue
Free Radical Biology and Medicine
Publisher
Elsevier BV
Published
2015-01
Volume
78
Pages
202-212
ISSN
0891-5849 (ISSN-L)
DOI
10.1016/j.freeradbiomed.2014.11.003
PMC
PMC4291150
OpenAlex
W1998845439
Cited by
232
References
27
Citations by year
2026: 15 · 2025: 15 · 2024: 23 · 2023: 11 · 2022: 34 · 2021: 26 · 2020: 24 · 2019: 17 · 2018: 20 · 2017: 25 · 2016: 14 · 2015: 7
FWCI
6.28
Type
journal article
Language
en
Open access
Licence
Locations
  • Free Radical Biology and Medicine (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • Medical Research Council
  • EMBO (ASTF 398-2013)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics, phytochemicals, and oxidative stress
  • Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Toxicology › Bioactive Compounds and Antitumor Agents
  • Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmacology › Drug-Induced Hepatotoxicity and Protection
Keywords
brusatol, Nrf2 inhibition, quassinoids, post-transcriptional regulation, hepatotoxicity, 1-chloro-2,4-dinitrobenzene, iodoacetamide, chemoresistance, hepatocytes, hepatoma cells, oxidative stress, drug safety
MeSH terms
Animals, Autophagy, Cells, Cultured, Gene Expression Regulation, Carcinoma, Hepatocellular, Humans, Liver Neoplasms, Oxidation-Reduction, RNA, Messenger, Blotting, Western, Signal Transduction, Apoptosis, Oxidative Stress, Reverse Transcriptase Polymerase Chain Reaction, Hepatocytes, Brucea, RNA, Small Interfering, Quassins, Cell Proliferation, NF-E2-Related Factor 2, Mice, Real-Time Polymerase Chain Reaction

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