Nrf2 Expression and Apoptosis in Quercetin-treated Malignant Mesothelioma Cells

Our plain-English summary of Nrf2 Expression and Apoptosis in Quercetin-treated Malignant Mesothelioma Cells, by Y.J. Lee, D.M. Lee, S.H. Lee (Molecules and Cells, 2015).

The study

• Authors: Y.J. Lee, D.M. Lee, S.H. Lee
• Journal: Molecules and Cells
• Published: 2015;38(5):416-25
• Cited by: 38 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

NF-E2-related factor 2 (Nrf2), a basic leucine zipper transcription factor, has recently received a great deal of attention as an important molecule that enhances antioxidative defenses and induces resistance to chemotherapy or radiotherapy. In this study, we investigated the apoptosis-inducing and Nrf2-upregulating effects of quercetin on malignant mesothelioma (MM) MSTO-211H and H2452 cells. Quercetin treatment inhibited cell growth and led to upregulation of Nrf2 at both the mRNA and protein levels without altering the ubiquitination and extending the half-life of the Nrf2 protein. Following treatment with quercetin, analyses of the nuclear level of Nrf2, Nrf2 antioxidant response element-binding assay, Nrf2 promoter-luc assay, and RT-PCR toward the Nrf2-regulated gene, heme oxygenase-1, demonstrated that the induced Nrf2 is transcriptionally active. Knockdown of Nrf2 expression with siRNA enhanced cytotoxicity due to the induction of apoptosis, as evidenced by an increase in the level of proapoptotic Bax, a decrease in the level of antiapoptotic Bcl-2 with enhanced cleavage of caspase-3 and PARP proteins, the appearance of a sub-G0/G1 peak in the flow cytometric assay, and increased percentage of apoptotic propensities in the annexin V binding assay. Effective reversal of apoptosis was observed following pretreatment with the pan-caspase inhibitor Z-VAD. Moreover, Nrf2 knockdown exhibited increased sensitivity to the anticancer drug, cisplatin, presumably by potentiating the oxidative stress induced by cisplatin. Collectively, our data demonstrate the importance of Nrf2 in cytoprotection, survival, and drug resistance with implications for the potential significance of targeting Nrf2 as a promising strategy for overcoming resistance to chemotherapeutics in MM.

In plain English

This laboratory study tested quercetin, a flavonoid found in fruits and vegetables, on two human malignant mesothelioma cell lines, MSTO-211H and H2452, grown in culture. No people or animals were studied, and asbestos was not used in the experiments. The authors focused on Nrf2, a protein that helps cells defend against oxidative stress. Quercetin lowered cell viability as the dose rose. At 20 micromolar it raised Nrf2 levels, and the authors' data suggested this came mainly from increased production of Nrf2 rather than slower breakdown. When Nrf2 expression was reduced with siRNA (knockdown), more cells died by apoptosis (programmed cell death). In the annexin V test, apoptotic cells reached 28.58% (MSTO-211H) and 25.54% (H2452) with Nrf2 knockdown plus quercetin, compared with 0.74% and 1% with control siRNA alone. A caspase inhibitor partly reversed this. Nrf2 knockdown also made the cells more sensitive to the chemotherapy drug cisplatin and raised cisplatin-induced reactive oxygen species. The authors concluded that targeting Nrf2 may be a promising strategy for overcoming chemotherapy resistance in mesothelioma. They called for further studies in animal models or humans.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory (in vitro) cell-culture experiment
• Who was studied: Two human malignant mesothelioma cell lines, MSTO-211H and H2452, obtained from the American Type Culture Collection; no human or animal subjects
• Exposure: Cells were treated with quercetin (0 to 100 micromolar; 20 micromolar in most experiments), had Nrf2 expression reduced by siRNA, and were treated with the chemotherapy drug cisplatin. Asbestos was not part of the experiments; the authors describe malignant mesothelioma as an asbestos-associated cancer.
• Main results:
  • In both cell lines, quercetin at 20 micromolar raised Nrf2 levels. The rise was first seen at 2 hours and lasted with longer incubation. At toxic doses of 60 micromolar and above, Nrf2 levels did not differ from untreated controls.
  • Quercetin did not lengthen the half-life of Nrf2 protein. The authors' data suggested the increase in Nrf2 occurred largely at the transcriptional level.
  • Nrf2-targeted siRNA repressed the transcript levels of Nrf2 and of the Nrf2-regulated gene HO-1, including the levels raised by quercetin.
  • Cell viability was compared with control siRNA alone. In MSTO-211H cells, viability fell to approximately 85.9% with Nrf2 siRNA alone and to 70.4% with Nrf2 siRNA plus 20 micromolar quercetin. In H2452 cells the corresponding figures were 88% and 73.8%.
  • In the annexin V apoptosis assay, the percentage of apoptotic cells was significantly elevated compared with control siRNA alone (0.74% in MSTO-211H, 1% in H2452). In MSTO-211H cells it was 18.68% with quercetin alone, 14.06% with Nrf2 siRNA alone and 28.58% with both combined. In H2452 cells the figures were 17.54%, 13.42% and 25.54%. The caspase inhibitor Z-VAD significantly, but not completely, restored these.
  • Cisplatin given for 72 hours inhibited viability, with an average IC50 of 76.1 micromolar in MSTO-211H cells. H2452 cells were much more resistant. Nrf2 knockdown significantly decreased cell survival compared with cisplatin after control siRNA.
  • Cisplatin at 80 micromolar for 24 hours increased reactive oxygen species from approximately 0.14% to 34% in MSTO-211H cells and from 0.4% to 9% in H2452 cells. With Nrf2 knockdown these rose to 45.9% and 33.7%, respectively.
• The authors' conclusion: The authors concluded that targeting Nrf2 is a promising strategy for inducing oxidative stress and killing mesothelioma cells. They saw it as a potential way to overcome resistance to some chemotherapeutic drugs in malignant mesothelioma.
• Limitations the authors state:
  • The authors stated that further studies are needed to clarify how quercetin can both induce cell death and favor cell protection through Nrf2 in mesothelioma cells.
  • The authors called for further assessment of efficacy and safety in animal models or human studies.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Mesothelioma.

Medical terms in this study, explained in our Lexicon: Apoptosis, Malignant Mesothelioma, Chemotherapy, Cisplatin, Oxidative Stress.

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