Adaptive response to GSH depletion and resistance to l-buthionine-(S,R)-sulfoximine: involvement of Nrf2 activation


Pharmacological depletion of l-γ-glutamyl-l-cysteinyl-glycine (GSH) has been implicated in the sensitization of cancer cells to alkylating agents and apoptosis. However, some types of cells do not induce apoptotic response following chemical depletion of GSH. In the present study, we report that murine embryonic fibroblasts (MEFs) can survive in the presence of GSH inhibitor l-buthionine-(S,R)-sulfoximine (BSO), even though most intracellular GSH was depleted. As a cellular adaptive mechanism, BSO treatment effectively activated the NF-E2-related factor 2 (Nrf2) pathway, which led to up-regulation of antioxidant enzymes in these cells through the extracellular signal-regulated kinase cascade. While nrf2-deficient MEFs lost the inducibility of antioxidant genes, which resulted in higher levels of reactive oxygen species accumulation, caspase-3 activation, and cell death than wild-type cells. Finally, nrf2-deficient cells can be more sensitized to doxorubicin-induced cell death by BSO pre-incubation, while wild-type cells were not. In addition, BSO-mediated cell death was facilitated by administering Nrf2 siRNA to chemoresistant human ovarian cancer cells. These results indicate that Nrf2 is the primary factor inducing the cell survival system under GSH depletion and that the effect of BSO as a chemosensitizer might be enhanced by inhibition of Nrf2.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6





Catalytic subunit of γ-glutamylcysteine ligase


Modulatory subunit of γ-glutamylcysteine ligase


NAD(P)H: quinone oxidoreductase 1


Antioxidant response element




Reactive oxygen speices


Mitogen-activated protein kinase


Extracellular signal-regulated kinase


c-Jun N-terminal kinase


  1. 1.

    Meister A, Anderson ME (1983) Glutathione. Annu Rev Biochem 52:711–760. doi:10.1146/

    PubMed  Article  CAS  Google Scholar 

  2. 2.

    Rahman I, Biswas SK, Jimenez LA, Torres M, Forman HJ (2005) Glutathione, stress responses, and redox signaling in lung inflammation. Antioxid Redox Signal 7:42–59. doi:10.1089/ars.2005.7.42

    PubMed  Article  CAS  Google Scholar 

  3. 3.

    Forman HJ, Dickinson DA (2003) Oxidative signaling and glutathione synthesis. Biofactors 17:1–12

    PubMed  CAS  Google Scholar 

  4. 4.

    Huang CS, Chang LS, Anderson ME, Meister A (1993) Catalytic and regulatory properties of the heavy subunit of rat kidney gamma-glutamylcysteine synthetase. J Biol Chem 268:19675–19680

    PubMed  CAS  Google Scholar 

  5. 5.

    Will O, Mahler HC, Arrigo AP, Epe B (1999) Influence of glutathione levels and heat-shock on the steady-state levels of oxidative DNA base modifications in mammalian cells. Carcinogenesis 20:333–337. doi:10.1093/carcin/20.2.333

    PubMed  Article  CAS  Google Scholar 

  6. 6.

    Walsh AC, Michaud SG, Malossi JA, Lawrence DA (1995) Glutathione depletion in human T lymphocytes: analysis of activation-associated gene expression and the stress response. Toxicol Appl Pharmacol 133:249–261. doi:10.1006/taap. 1995.1149

    PubMed  Article  CAS  Google Scholar 

  7. 7.

    Kensler TW, Wakabayashi N, Biswal S (2007) Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway. Annu Rev Pharmacol Toxicol 47:89–116. doi:10.1146/annurev.pharmtox.46.120604.141046

    PubMed  Article  CAS  Google Scholar 

  8. 8.

    Kobayashi M, Yamamoto M (2005) Molecular mechanisms activating the Nrf2-Keap1 pathway of antioxidant gene regulation. Antioxid Redox Signal 7:385–394. doi:10.1089/ars.2005.7.385

    PubMed  Article  CAS  Google Scholar 

  9. 9.

    Wild AC, Moinova HR, Mulcahy RT (1999) Regulation of gamma-glutamylcysteine synthetase subunit gene expression by the transcription factor Nrf2. J Biol Chem 274:33627–33636. doi:10.1074/jbc.274.47.33627

    PubMed  Article  CAS  Google Scholar 

  10. 10.

    Moinova HR, Mulcahy RT (1999) Up-regulation of the human gamma-glutamylcysteine synthetase regulatory subunit gene involves binding of Nrf-2 to an electrophile responsive element. Biochem Biophys Res Commun 261:661–668. doi:10.1006/bbrc.1999.1109

    PubMed  Article  CAS  Google Scholar 

  11. 11.

    Zucker B, Hanusch J, Bauer G (1997) Glutathione depletion in fibroblasts is the basis for apoptosis-induction by endogenous reactive oxygen species. Cell Death Differ 4:388–395. doi:10.1038/sj.cdd.4400258

    PubMed  Article  CAS  Google Scholar 

  12. 12.

    Anderson CP, Tsai J, Chan W, Park CK, Tian L, Lui RM et al (1997) Buthionine sulphoximine alone and in combination with melphalan (l-PAM) is highly cytotoxic for human neuroblastoma cell lines. Eur J Cancer 33:2016–2019. doi:10.1016/S0959-8049(97)00203-7

    PubMed  Article  CAS  Google Scholar 

  13. 13.

    Sugimoto C, Matsukawa S, Fujieda S, Noda I, Tanaka N, Tsuzuki H et al (1996) Involvement of intracellular glutathione in induction of apoptosis by cisplatin in a human pharyngeal carcinoma cell line. Anticancer Res 16:675–680

    PubMed  CAS  Google Scholar 

  14. 14.

    Voehringer DW (1999) BCL-2 and glutathione: alterations in cellular redox state that regulate apoptosis sensitivity. Free Radic Biol Med 27:945–950. doi:10.1016/S0891-5849(99)00174-4

    PubMed  Article  CAS  Google Scholar 

  15. 15.

    Kwak MK, Wakabayashi N, Greenlaw JL, Yamamoto M, Kensler TW (2003) Antioxidants enhance mammalian proteasome expression through the Keap1-Nrf2 signaling pathway. Mol Cell Biol 23:8786–8794. doi:10.1128/MCB.23.23.8786-8794.2003

    PubMed  Article  CAS  Google Scholar 

  16. 16.

    Griffith OW (1980) Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal Biochem 106:207–212. doi:10.1016/0003-2697(80)90139-6

    PubMed  Article  CAS  Google Scholar 

  17. 17.

    Carmichael J, DeGraff WG, Gazdar AF, Minna JD, Mitchell JB (1987) Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res 47:936–942

    PubMed  CAS  Google Scholar 

  18. 18.

    Cho J-M, Manandhar S, Lee H-R, Park H-M, Kwak M-K (2008) Role of the Nrf2-antioxidant system in cytotoxicity mediated by anticancer cisplatin: implication to cancer cell resistance. Cancer Lett 260:96–108. doi:10.1016/j.canlet.2007.10.022

    PubMed  Article  CAS  Google Scholar 

  19. 19.

    Jenner P, Olanow CW (1996) Oxidative stress and the pathogenesis of Parkinson’s disease. Neurology 47:S161–S170

    PubMed  CAS  Google Scholar 

  20. 20.

    Cecchi C, Latorraca S, Sorbi S, Iantomasi T, Favilli F, Vincenzini MT et al (1999) Glutathione level is altered in lymphoblasts from patients with familial Alzheimer’s disease. Neurosci Lett 275:152–154. doi:10.1016/S0304-3940(99)00751-X

    PubMed  Article  CAS  Google Scholar 

  21. 21.

    Kwak MK, Wakabayashi N, Itoh K, Motohashi H, Yamamoto M, Kensler TW (2003) Modulation of gene expression by cancer chemopreventive dithiolethiones through the Keap1-Nrf2 pathway. Identification of novel gene clusters for cell survival. J Biol Chem 278:8135–8145. doi:10.1074/jbc.M211898200

    PubMed  Article  CAS  Google Scholar 

  22. 22.

    Cho HY, Reddy SP, Debiase A, Yamamoto M, Kleeberger SR (2005) Gene expression profiling of NRF2-mediated protection against oxidative injury. Free Radic Biol Med 38:325–343. doi:10.1016/j.freeradbiomed.2004.10.013

    PubMed  Article  CAS  Google Scholar 

  23. 23.

    Filomeni G, Aquilano K, Rotilio G, Ciriolo MR (2005) Antiapoptotic response to induced GSH depletion: involvement of heat shock proteins and NF-kappaB activation. Antioxid Redox Signal 7:446–455. doi:10.1089/ars.2005.7.446

    PubMed  Article  CAS  Google Scholar 

  24. 24.

    Rahman I, MacNee W (2000) Regulation of redox glutathione levels and gene transcription in lung inflammation: therapeutic approaches. Free Radic Biol Med 28:1405–1420. doi:10.1016/S0891-5849(00)00215-X

    PubMed  Article  CAS  Google Scholar 

  25. 25.

    D’Alessio M, Cerella C, Amici C, Pesce C, Coppola S, Fanelli C et al (2004) Glutathione depletion up-regulates Bcl-2 in BSO-resistant cells. FASEB J 18:1609–1611

    PubMed  CAS  Google Scholar 

  26. 26.

    Vahrmeijer AL, Hoetelmans RW, Mulder GJ, Schutrups J, van Vlierberghe RL, van de Velde CJ et al (2000) Development of resistance to glutathione depletion-induced cell death in CC531 colon carcinoma cells: association with increased expression of bcl-2. Biochem Pharmacol 59:1557–1562. doi:10.1016/S0006-2952(00)00286-0

    PubMed  Article  CAS  Google Scholar 

  27. 27.

    Dinkova-Kostova AT, Holtzclaw WD, Cole RN, Itoh K, Wakabayashi N, Katoh Y et al (2002) Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants. Proc Natl Acad Sci USA 99:11908–11913. doi:10.1073/pnas.172398899

    PubMed  Article  CAS  Google Scholar 

  28. 28.

    Wakabayashi N, Dinkova-Kostova AT, Holtzclaw WD, Kang ML, Kobayashi A, Yamamoto M et al (2004) Protection against electrophile and oxidant stress by induction of the phase 2 response: fate of cysteines of the Keap1 sensor modified by inducers. Proc Natl Acad Sci USA 101:2040–2045. doi:10.1073/pnas.0307301101

    PubMed  Article  CAS  Google Scholar 

  29. 29.

    Kang KW, Cho MK, Lee CH, Kim SG (2001) Activation of phosphatidylinositol 3-kinase and Akt by tert-butylhydroquinone is responsible for antioxidant response element-mediated rGSTA2 induction in H4IIE cells. Mol Pharmacol 59:1147–1156

    PubMed  CAS  Google Scholar 

  30. 30.

    Yu R, Chen C, Mo YY, Hebbar V, Owuor ED, Tan TH et al (2000) Activation of mitogen-activated protein kinase pathways induces antioxidant response element-mediated gene expression via a Nrf2-dependent mechanism. J Biol Chem 275:39907–39913. doi:10.1074/jbc.M004037200

    PubMed  Article  CAS  Google Scholar 

  31. 31.

    Matsuzawa A, Ichijo H (2005) Stress-responsive protein kinases in redox-regulated apoptosis signaling. Antioxid Redox Signal 7:472–481. doi:10.1089/ars.2005.7.472

    PubMed  Article  CAS  Google Scholar 

Download references


This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MOST, R01-2007-000-10890-0)

Author information



Corresponding author

Correspondence to Mi-Kyoung Kwak.

Additional information

Hyang-Rim Lee and Jeong-Min Cho contributed equally to this work.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Lee, H., Cho, J., Shin, D. et al. Adaptive response to GSH depletion and resistance to l-buthionine-(S,R)-sulfoximine: involvement of Nrf2 activation. Mol Cell Biochem 318, 23–31 (2008).

Download citation


  • GSH
  • BSO
  • Oxidative stress
  • Nrf2
  • Chemosensitizer