BAP1 Downregulates NRF2 Target Genes and Exerts Anti-Tumorigenic Effects by Deubiquitinating KEAP1 in Lung Adenocarcinoma.

KELCH-ECH-associated protein 1 (KEAP1) is an adaptor protein of Cullin 3 (CUL3) E3 ubiquitin ligase that targets a redox sensitive transcription factor, NF-E2-related factor 2 (NRF2). BRCA1-associated protein 1 (BAP1) is a tumor suppressor and deubiquitinase whose mutations increase the risk of several types of familial cancers. In the present study, we have identified that BAP1 deubiquitinates KEAP1 by binding to the BTB domain. Lentiviral transduction of BAP1 decreased the expression of NRF2 target genes, suppressed the migration and invasion, and sensitized cisplatin-induced apoptosis in human lung adenocarcinoma (LUAD) A549 cells. Examination of the lung tissues in KrasG12D/+ mice demonstrated that the level of Bap1 and Keap1 mRNAs progressively decreases during lung tumor progression, and it is correlated with NRF2 activation and the inhibition of oxidative stress. Supporting this observation, lentiviral transduction of BAP1 decreased the growth of A549 xenografts in athymic nude mice. Transcriptome analysis of human lung tissues showed that the levels of Bap1 mRNA are significantly higher in normal samples than LUAD samples. Moreover, the expression of Bap1 mRNA is associated with a better survival of LUAD patients. Together, our study demonstrates that KEAP1 deubiquitination by BAP1 is novel tumor suppressive mechanism of LUAD.

Cardamonin Inhibits Oxazolone-Induced Atopic Dermatitis by the Induction of NRF2 and the Inhibition of Th2 Cytokine Production.

The skin is constantly exposed to various types of chemical stresses that challenge the immune cells, leading to the activation of T cell-mediated hypersensitivity reactions including atopic dermatitis. Previous studies have demonstrated that a variety of natural compounds are effective against development of atopic dermatitis by modulating immune responses. Cardamonin is a natural compound abundantly found in cardamom spices and many other medicinal plant species. In the present study, we attempted to examine whether cardamonin could inhibit oxazolone-induced atopic dermatitis in vivo. Our results show that topical application of cardamonin onto the ear of mice suppressed oxazolone-induced inflammation in the ear and hyperplasia in the spleen. Cardamonin also inhibited oxazolone-induced destruction of connective tissues and subsequent infiltration of mast cells into the skin. In addition, we found that the production of Th2 cytokines is negatively regulated by NRF2, and the induction of NRF2 by cardamonin contributed to suppressing oxazolone-induced Th2 cytokine production and oxidative damages in vivo. Together, our results demonstrate that cardamonin is a promising natural compound, which might be effective for treatment of atopic dermatitis.

Molecular mechanisms and systemic targeting of NRF2 dysregulation in cancer.

NF-E2-related factor 2 (NRF2) is a master regulator of redox homeostasis and provides cellular protection against oxidants and electrophiles by inducing the expression of a wide array of phase II cytoprotective genes. Until now, a number of NRF2 activators have been developed for treatment of chronic diseases and some are under evaluation in the clinical studies. On the other hand, accumulating evidence indicates that NRF2 confers chemoresistance and radioresistance, and its expression is correlated with poor prognosis in cancer patients. Studies in the last decade demonstrate that diverse mechanisms such as somatic mutations, accumulation of KEAP1 binding proteins, transcriptional dysregulation, oncogene activation, and accumulation of reactive metabolites contribute to NRF2 activation in cancer. In the present review, we illustrate the molecular mechanisms governing the function of NRF2 and explain how they are hijacked in cancer. We also provide some examples of NRF2 inhibitors together with a brief explanation of their mechanisms of action.

Homoharringtonine stabilizes secondary structure of guanine-rich sequence existing in the 5'-untranslated region of Nrf2.

Homoharringtonine, known as omacetaxine mepesuccinate, is a pharmaceutical drug substance approved for treatment of chronic myeloid leukemia. Here, we report that homoharringtonine (HHT) is a novel chemical inhibitor of NRF2. HHT significantly suppressed NRF2 and ARE-dependent gene expression in human lung carcinoma A549 cells. HHT stabilized secondary structure of guanine-rich sequence existing in the 5'-untranslated region (5'-UTR) of Nrf2 and sensitized A549 cells to etoposide-induced apoptosis. To the best of our knowledge, HHT is the first type of transcriptional inhibitor of Nrf2 that stabilizes guanine-rich sequence existing in the 5'-UTR. Our study also provides a novel mechanism of action underlying how HHT exerts anti-carcinogenic effects in cancer cells.

4'-O-ß-D-Glucosyl-5-O-Methylvisamminol Attenuates Pro-Inflammatory Responses and Protects against Oxidative Damages.

We attempted to examine anti-inflammatory and anti-oxidant effects of 4'-O-ß-D-glucosyl-5-O-methylvisamminol (GOMV), the first epigenetic inhibitor of histone phosphorylation at Ser10. While GOMV did not affect the viability of murine macrophage RAW 264.7 cells, it significantly suppressed lipopolysaccharide (LPS)-induced generation of prostaglandin E2 (PGE2) and nitric oxide (NO) through transcriptional inhibition of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). GOMV also scavenged free radicals in vitro, increased NF-E2-related factor 2 (NRF2), and activated antioxidant response element (ARE), thereby resulting in the induction of phase II cytoprotective enzymes in human keratinocyte HaCaT cells. Finally, GOMV significantly protected HaCaT cells against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced oxidative intracellular damages. Together, our results illustrate that GOMV possesses anti-inflammatory and anti-oxidant activity.

Suppression of NRF2/ARE by convallatoxin sensitises A549 cells to 5-FU-mediated apoptosis.

NF-E2-related factor 2 (NRF2) regulates transcription of phase II cytoprotective enzymes to protect normal cells against oxidative stress. However, a high level of NRF2 offers a growth advantage, chemoresistance, and radioresistance in cancer. In the present study, we have identified convallatoxin as a novel inhibitor of NRF2/ARE. Suppression of NRF2 by convallatoxin was not transcriptionally mediated, but regulated at the level of proteolysis. Convallatoxin activated GSK-3ß and suppression of NRF2 by convallatoxin required the Neh6 domain. Convallatoxin sensitised A549 cells to 5-fluorouracil-mediated cell death by promoting apoptosis. Together, our results provide evidence that convallatoxin might be useful as a chemotherapeutic adjuvant due to its ability to suppress NRF2/ARE.

Marliolide inhibits skin carcinogenesis by activating NRF2/ARE to induce heme oxygenase-1.

Heme oxygenase-1 (HO-1) catalyzes the enzymatic degradation of heme to produce three anti-oxidant molecules: carbon monoxide (CO), ferrous ion (Fe2+), and biliverdin. Induction of HO-1 is currently considered as a feasible strategy to treat oxidative stress-related diseases. In the present study, we identified marliolide as a novel inducer of HO-1 in human normal keratinocyte HaCaT cells. Mechanism-based studies demonstrated that the induction of HO-1 by marliolide occurred through activation of NRF2/ARE via direct binding of marliolide to KEAP1. Structure-activity relationship revealed chemical moieties of marliolide critical for induction of HO-1, which renders a support for Michael reaction as a potential mechanism of action. Finally, we observed that marliolide significantly inhibited the papilloma formation in DMBA/TPA-induced mouse skin carcinogenesis model and this event was closely associated with lowering the formation of 8-OH-G and 4-HNE in vivo. Together, our study provides the first evidence that marliolide might be effective against oxidative stress-related skin disorders.

Ethanol Extract of Cirsium japonicum var. ussuriense Kitamura Exhibits the Activation of Nuclear Factor Erythroid 2-Related Factor 2-dependent Antioxidant Response Element and Protects Human Keratinocyte HaCaT Cells Against Oxidative DNA Damage.

Keratinocytes are constantly exposed to extracellular insults, such as ultraviolet B, toxic chemicals and mechanical stress, all of which can facilitate the aging of keratinocytes via the generation of intracellular reactive oxygen species (ROS). Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that plays a critical role in protecting keratinocytes against oxidants and xenobiotics by binding to the antioxidant response element (ARE), a cis-acting element existing in the promoter of most phase II cytoprotective genes. In the present study, we have attempted to find novel ethanol extract(s) of indigenous plants of Jeju island, Korea that can activate the Nrf2/ARE-dependent gene expression in human keratinocyte HaCaT cells. As a result, we identified that ethanol extract of Cirsium japonicum var. ussuriense Kitamura (ECJUK) elicited strong stimulatory effect on the ARE-dependent gene expression. Supporting this observation, we found that ECJUK induced the expression of Nrf2, hemoxygenase-1, and NAD(P)H: quinone oxidoreductase-1 and this event was correlated with Akt1 phosphorylation. We also found that ECJUK increased the intracellular reduced glutathione level and suppressed 12-O-tetradecanoylphorbol acetate-induced 8-hydroxyguanosine formation without affecting the overall viability. Collectively, our results provide evidence that ECJUK can protect against oxidative stress-mediated damages through the activation of Nrf2/ARE-dependent phase II cytoprotective gene expression.