Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Inhibition of the Nrf2 transcription factor by the alkaloid trigonelline renders pancreatic cancer cells more susceptible to apoptosis through decreased proteasomal gene expression and proteasome activity

Oncogene volume 32, pages 4825–4835 (2013)Cite this article

Subjects

Abstract

Evidence accumulates that the transcription factor nuclear factor E2-related factor 2 (Nrf2) has an essential role in cancer development and chemoresistance, thus pointing to its potential as an anticancer target and undermining its suitability in chemoprevention. Through the induction of cytoprotective and proteasomal genes, Nrf2 confers apoptosis protection in tumor cells, and inhibiting Nrf2 would therefore be an efficient strategy in anticancer therapy. In the present study, pancreatic carcinoma cell lines (Panc1, Colo357 and MiaPaca2) and H6c7 pancreatic duct cells were analyzed for the Nrf2-inhibitory effect of the coffee alkaloid trigonelline (trig), as well as for its impact on Nrf2-dependent proteasome activity and resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and anticancer drug-induced apoptosis. Chemoresistant Panc1 and Colo357 cells exhibit high constitutive Nrf2 activity, whereas chemosensitive MiaPaca2 and H6c7 cells display little basal but strong tert-butylhydroquinone (tBHQ)-inducible Nrf2 activity and drug resistance. Trig efficiently decreased basal and tBHQ-induced Nrf2 activity in all cell lines, an effect relying on a reduced nuclear accumulation of the Nrf2 protein. Along with Nrf2 inhibition, trig blocked the Nrf2-dependent expression of proteasomal genes (for example, s5a/psmd4 and α5/psma5) and reduced proteasome activity in all cell lines tested. These blocking effects were absent after treatment with Nrf2 siRNA, a condition in which proteasomal gene expression and proteasome activity were already decreased, whereas siRNA against the related transcription factor Nrf1 did not affect proteasome activity and the inhibitory effect of trig. Depending on both Nrf2 and proteasomal gene expression, the sensitivity of all cell lines to anticancer drugs and TRAIL-induced apoptosis was enhanced by trig. Moreover, greater antitumor responses toward anticancer drug treatment were observed in tumor-bearing mice when receiving trig. In conclusion, representing an efficient Nrf2 inhibitor capable of blocking Nrf2-dependent proteasome activity and thereby apoptosis protection in pancreatic cancer cells, trig might be beneficial in improving anticancer therapy.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8

Similar content being viewed by others

Abbreviations

ARE:

antioxidant response element

LMB:

leptomycin-B

Nrf2 & -1:

nuclear factor E2-related factor 2 & -1

PARP1:

poly(ADP-ribose)–polymerase 1

PDAC:

pancreatic ductal adenocarcinoma

SCID:

severe combined immunodeficiency

Suc-LLVY-AMC:

N-succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosyl-7-amido-4-methylcumarin

tBHQ:

tertbutylhydroxyquinone

TRAIL:

tumor necrosis factor-related apoptosis-inducing ligand

trig:

trigonelline

References

  1. Arlt A, Bauer I, Schafmayer C, Tepel J, Muerkoster SS, Brosch M et al. Increased proteasome subunit protein expression and proteasome activity in colon cancer relate to an enhanced activation of nuclear factor E2-related factor 2 (Nrf2). Oncogene 2009; 28: 3983–3996.

    Article  CAS  Google Scholar 

  2. Akhdar H, Loyer P, Rauch C, Corlu A, Guillouzo A, Morel F . Involvement of Nrf2 activation in resistance to 5-fluorouracil in human colon cancer HT-29 cells. Eur J Cancer 2009; 45: 2219–2227.

    Article  CAS  Google Scholar 

  3. DeNicola GM, Karreth FA, Humpton TJ, Gopinathan A, Wei C, Frese K et al. Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis. Nature 2011; 475: 106–109.

    Article  CAS  Google Scholar 

  4. Hong YB, Kang HJ, Kwon SY, Kim HJ, Kwon KY, Cho CH et al. Nuclear factor (erythroid-derived 2)-like 2 regulates drug resistance in pancreatic cancer cells. Pancreas 2010; 39: 463–472.

    Article  CAS  Google Scholar 

  5. Jiang T, Chen N, Zhao F, Wang XJ, Kong B, Zheng W et al. High levels of Nrf2 determine chemoresistance in type II endometrial cancer. Cancer Res 2010; 70: 5486–5496.

    Article  CAS  Google Scholar 

  6. Kim TH, Hur EG, Kang SJ, Kim JA, Thapa D, Lee YM et al. NRF2 blockade suppresses colon tumor angiogenesis by inhibiting hypoxia-induced activation of HIF-1alpha. Cancer Res 2011; 71: 2260–2275.

    Article  CAS  Google Scholar 

  7. Kim SK, Yang JW, Kim MR, Roh SH, Kim HG, Lee KY et al. Increased expression of Nrf2/ARE-dependent anti-oxidant proteins in tamoxifen-resistant breast cancer cells. Free Radic Biol Med 2008; 45: 537–546.

    Article  CAS  Google Scholar 

  8. Konstantinopoulos PA, Spentzos D, Fountzilas E, Francoeur N, Sanisetty S, Grammatikos AP et al. Keap1 mutations and Nrf2 pathway activation in epithelial ovarian cancer. Cancer Res 2011; 71: 5081–5089.

    Article  CAS  Google Scholar 

  9. Lister A, Nedjadi T, Kitteringham NR, Campbell F, Costello E, Lloyd B et al. Nrf2 is overexpressed in pancreatic cancer: implications for cell proliferation and therapy. Mol Cancer 2011; 10: 37.

    Article  CAS  Google Scholar 

  10. Singh A, Misra V, Thimmulappa RK, Lee H, Ames S, Hoque MO et al. Dysfunctional KEAP1-NRF2 interaction in non-small-cell lung cancer. PLoS Med 2006; 3: e420.

    Article  Google Scholar 

  11. Solis LM, Behrens C, Dong W, Suraokar M, Ozburn NC, Moran CA et al. Nrf2 and Keap1 abnormalities in non-small cell lung carcinoma and association with clinicopathologic features. Clin Cancer Res 2010; 16: 3743–3753.

    Article  CAS  Google Scholar 

  12. Hu Y, Ju Y, Lin D, Wang Z, Huang Y, Zhang S et al. Mutation of the Nrf2 gene in non-small cell lung cancer. Mol Biol Rep 2012; 39: 4743–4747.

    Article  CAS  Google Scholar 

  13. Kim YR, Oh JE, Kim MS, Kang MR, Park SW, Han JY et al. Oncogenic NRF2 mutations in squamous cell carcinomas of oesophagus and skin. J Pathol 2010; 220: 446–451.

    Article  CAS  Google Scholar 

  14. Wang R, An J, Ji F, Jiao H, Sun H, Zhou D . Hypermethylation of the Keap1 gene in human lung cancer cell lines and lung cancer tissues. Biochem Biophys Res Commun 2008; 373: 151–154.

    Article  CAS  Google Scholar 

  15. Eades G, Yang M, Yao Y, Zhang Y, Zhou Q . miR-200a regulates Nrf2 activation by targeting Keap1 mRNA in breast cancer cells. J Biol Chem 2011; 286: 40725–40733.

    Article  CAS  Google Scholar 

  16. Nioi P, Nguyen T . A mutation of Keap1 found in breast cancer impairs its ability to repress Nrf2 activity. Biochem Biophys Res Commun 2007; 362: 816–821.

    Article  CAS  Google Scholar 

  17. Kinch L, Grishin NV, Brugarolas J . Succination of Keap1 and activation of Nrf2-dependent antioxidant pathways in FH-deficient papillary renal cell carcinoma type 2. Cancer Cell 2011; 20: 418–420.

    Article  CAS  Google Scholar 

  18. Adam J, Hatipoglu E, O'Flaherty L, Ternette N, Sahgal N, Lockstone H et al. Renal cyst formation in Fh1-deficient mice is independent of the Hif/Phd pathway: roles for fumarate in KEAP1 succination and Nrf2 signaling. Cancer Cell 2011; 20: 524–537.

    Article  CAS  Google Scholar 

  19. Kim J, Cha YN, Surh YJ . A protective role of nuclear factor-erythroid 2-related factor-2 (Nrf2) in inflammatory disorders. Mutat Res 2010; 690: 12–23.

    Article  CAS  Google Scholar 

  20. Sebens S, Bauer I, Geismann C, Grage-Griebenow E, Ehlers S, Kruse ML et al. Inflammatory macrophages induce NRF2 dependent proteasome activity in colonic NCM460 cells and thereby confer anti-apoptotic protection. J Biol Chem 2011; 286: 40911–40921.

    Article  CAS  Google Scholar 

  21. Singh S, Vrishni S, Singh BK, Rahman I, Kakkar P . Nrf2-ARE stress response mechanism: a control point in oxidative stress-mediated dysfunctions and chronic inflammatory diseases. Free Radic Res 2010; 44: 1267–1288.

    Article  CAS  Google Scholar 

  22. Du ZX, Yan Y, Zhang HY, Liu BQ, Gao YY, Niu XF et al. Proteasome inhibition induces a p38 MAPK pathway-dependent antiapoptotic program via Nrf2 in thyroid cancer cells. J Clin Endocrinol Metab 2011; 96: E763–E771.

    Article  CAS  Google Scholar 

  23. Singh A, Bodas M, Wakabayashi N, Bunz F, Biswal S . Gain of Nrf2 function in non-small-cell lung cancer cells confers radioresistance. Antioxid Redox Signal 2010; 13: 1627–1637.

    Article  CAS  Google Scholar 

  24. Shim GS, Manandhar S, Shin DH, Kim TH, Kwak MK . Acquisition of doxorubicin resistance in ovarian carcinoma cells accompanies activation of the NRF2 pathway. Free Radic Biol Med 2009; 47: 1619–1631.

    Article  CAS  Google Scholar 

  25. Zhang DD . The Nrf2-Keap1-ARE signaling pathway: the regulation and dual function of Nrf2 in cancer. Antioxid Redox Signal 2010; 13: 1623–1626.

    Article  CAS  Google Scholar 

  26. Kim TH, Hur EG, Kang SJ, Kim JA, Thapa D, Lee YM et al. NRF2 blockade suppresses colon tumor angiogenesis by inhibiting hypoxia-induced activation of HIF-1α. Cancer Res 2011; 71: 2260–2275.

    Article  CAS  Google Scholar 

  27. Chen L, Madura K . Increased proteasome activity, ubiquitin-conjugating enzymes, and eEF1A translation factor detected in breast cancer tissue. Cancer Res 2005; 65: 5599–5606.

    Article  CAS  Google Scholar 

  28. Hu XT, Chen W, Zhang FB, Shi QL, Hu JB, Geng SM et al. Depletion of the proteasome subunit PSMA7 inhibits colorectal cancer cell tumorigenicity and migration. Oncol Rep 2009; 22: 1247–1252.

    CAS  PubMed  Google Scholar 

  29. Ren S, Smith MJ, Louro ID, McKie-Bell P, Bani MR, Wagner M et al. The p44S10 locus, encoding a subunit of the proteasome regulatory particle, is amplified during progression of cutaneous malignant melanoma. Oncogene 2000; 19: 1419–1427.

    Article  CAS  Google Scholar 

  30. Rho JH, Qin S, Wang JY, Roehrl MH . Proteomic expression analysis of surgical human colorectal cancer tissues: up-regulation of PSB7, PRDX1, and SRP9 and hypoxic adaptation in cancer. J Proteome Res 2008; 7: 2959–2972.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  32. Kwak MK, Wakabayashi N, Greenlaw JL, Yamamoto M, Kensler TW . Antioxidants enhance mammalian proteasome expression through the Keap1-Nrf2 signaling pathway. Mol Cell Biol 2003; 23: 8786–8794.

    Article  CAS  Google Scholar 

  33. Kapeta S, Chondrogianni N, Gonos ES . Nuclear erythroid factor 2-mediated proteasome activation delays senescence in human fibroblasts. J Biol Chem 2010; 285: 8171–8184.

    Article  CAS  Google Scholar 

  34. Schaedler S, Krause J, Himmelsbach K, Carvajal-Yepes M, Lieder F, Klingel K et al. Hepatitis B virus induces expression of antioxidant response element-regulated genes by activation of Nrf2. J Biol Chem 2010; 285: 41074–41086.

    Article  CAS  Google Scholar 

  35. Kwak MK, Kensler TW . Induction of 26S proteasome subunit PSMB5 by the bifunctional inducer 3-methylcholanthrene through the Nrf2-ARE, but not the AhR/Arnt-XRE, pathway. Biochem Biophys Res Commun 2006; 345: 1350–1357.

    Article  CAS  Google Scholar 

  36. Kwak MK, Itoh K, Yamamoto M, Kensler TW . Enhanced expression of the transcription factor Nrf2 by cancer chemopreventive agents: role of antioxidant response element-like sequences in the nrf2 promoter. Mol Cell Biol 2002; 22: 2883–2892.

    Article  CAS  Google Scholar 

  37. Lau A, Villeneuve NF, Sun Z, Wong PK, Zhang DD . Dual roles of Nrf2 in cancer. Pharmacol Res 2008; 58: 262–270.

    Article  CAS  Google Scholar 

  38. Reuter S, Gupta SC, Chaturvedi MM, Aggarwal BB . Oxidative stress, inflammation, and cancer: how are they linked? Free Radic Biol Med 2010; 49: 1603–1616.

    Article  CAS  Google Scholar 

  39. Taguchi A, Politi K, Pitteri SJ, Lockwood WW, Faca VM, Kelly-Spratt K et al. Lung cancer signatures in plasma based on proteome profiling of mouse tumor models. Cancer Cell 2011; 20: 289–299.

    Article  CAS  Google Scholar 

  40. Hayes JD, McMahon M . NRF2 and KEAP1 mutations: permanent activation of an adaptive response in cancer. Trends Biochem Sci 2009; 34: 176–188.

    Article  CAS  Google Scholar 

  41. Osburn WO, Kensler TW . Nrf2 signaling: an adaptive response pathway for protection against environmental toxic insults. Mutat Res 2008; 659: 31–39.

    Article  CAS  Google Scholar 

  42. Wang XJ, Sun Z, Villeneuve NF, Zhang S, Zhao F, Li Y et al. Nrf2 enhances resistance of cancer cells to chemotherapeutic drugs, the dark side of Nrf2. Carcinogenesis 2008; 29: 1235–1243.

    Article  CAS  Google Scholar 

  43. Wang XJ, Hayes JD, Henderson CJ, Wolf CR . Identification of retinoic acid as an inhibitor of transcription factor Nrf2 through activation of retinoic acid receptor alpha. Proc Natl Acad Sci USA 2007; 104: 19589–19594.

    Article  CAS  Google Scholar 

  44. Zhou W, Lo SC, Liu JH, Hannink M, Lubahn DB . ERRbeta: a potent inhibitor of Nrf2 transcriptional activity. Mol Cell Endocrinol 2007; 278: 52–62.

    Article  CAS  Google Scholar 

  45. Boettler U, Sommerfeld K, Volz N, Pahlke G, Teller N, Somoza V et al. Coffee constituents as modulators of Nrf2 nuclear translocation and ARE (EpRE)-dependent gene expression. J Nutr Biochem 2011; 22: 426–440.

    Article  CAS  Google Scholar 

  46. Kannan S, Jaiswal AK . Low and high dose UVB regulation of transcription factor NF-E2-related factor 2. Cancer Res 2006; 66: 8421–8429.

    Article  CAS  Google Scholar 

  47. Steffen J, Seeger M, Koch A, Kruger E . Proteasomal degradation is transcriptionally controlled by TCF11 via an ERAD-dependent feedback loop. Mol Cell 2010; 40: 147–158.

    Article  CAS  Google Scholar 

  48. Arlt A, Vorndamm J, Breitenbroich M, Folsch UR, Kalthoff H, Schmidt WE et al. Inhibition of NF-kappaB sensitizes human pancreatic carcinoma cells to apoptosis induced by etoposide (VP16) or doxorubicin. Oncogene 2001; 20: 859–868.

    Article  CAS  Google Scholar 

  49. Fritsche P, Seidler B, Schuler S, Schnieke A, Gottlicher M, Schmid RM et al. HDAC2 mediates therapeutic resistance of pancreatic cancer cells via the BH3-only protein NOXA. Gut 2009; 58: 1399–1409.

    Article  CAS  Google Scholar 

  50. Stathis A, Moore MJ . Advanced pancreatic carcinoma: current treatment and future challenges. Nat Rev 2010; 7: 163–172.

    CAS  Google Scholar 

  51. Elangovan M, Oh C, Sukumaran L, Wojcik C, Yoo YJ . Functional differences between two major ubiquitin receptors in the proteasome; S5a and hRpn13. Biochem Biophys Res Commun 2010; 396: 425–428.

    Article  CAS  Google Scholar 

  52. Nguyen T, Sherratt PJ, Nioi P, Yang CS, Pickett CB . Nrf2 controls constitutive and inducible expression of ARE-driven genes through a dynamic pathway involving nucleocytoplasmic shuttling by Keap1. J Biol Chem 2005; 280: 32485–32492.

    Article  CAS  Google Scholar 

  53. Niture SK, Jaiswal AK . Prothymosin-α mediates nuclear import of the INrf2/Cul3_Rbx1 complex to degrade nuclear Nrf2. J Biol Chem 2009; 284: 13856–13868.

    Article  CAS  Google Scholar 

  54. Radhakrishnan SK, Lee CS, Young P, Beskow A, Chan JY, Deshaies RJ . Transcription factor Nrf1 mediates the proteasome recovery pathway after proteasome inhibition in mammalian cells. Mol Cell 2010; 38: 17–28.

    Article  CAS  Google Scholar 

  55. Xie Y . Feedback regulation of proteasome gene expression and its implications in cancer therapy. Cancer Metastasis Rev 2010; 29: 687–693.

    Article  CAS  Google Scholar 

  56. Nguyen T, Nioi P, Pickett CB . The Nrf2-antioxidant response element signaling pathway and its activation by oxidative stress. J Biol Chem 2009; 284: 13291–13295.

    Article  CAS  Google Scholar 

  57. van Dijk AE, Olthof MR, Meeuse JC, Seebus E, Heine RJ, van Dam RM . Acute effects of decaffeinated coffee and the major coffee components chlorogenic acid and trigonelline on glucose tolerance. Diabetes Care 2009; 32: 1023–1025.

    Article  CAS  Google Scholar 

  58. Olthof MR, van Dijk AE, Deacon CF, Heine RJ, van Dam RM . Acute effects of decaffeinated coffee and the major coffee components chlorogenic acid and trigonelline on incretin hormones. Nutr Metab (Lond) 2011; 8: 10.

    Article  CAS  Google Scholar 

  59. Geismann C, Arlt A, Bauer I, Pfeifer M, Schirmer U, Altevogt P et al. Binding of the transcription factor slug to the L1CAM promoter is essential for transforming growth factor-beta1 (TGF-beta)-induced L1CAM expression in human pancreatic ductal adenocarcinoma cells. Int J Oncol 2011; 38: 257–266.

    CAS  Google Scholar 

  60. Müerköster S, Arlt A, Sipos B, Witt M, Großmann M, Klöppel G et al. Increased expression of the E3-ubiquitin ligase receptor subunit βTRCP1 relates to constitutive NF-κB activation and chemoresistance in pancreatic carcinoma cells. Cancer Res 2005; 65: 1316–1324.

    Article  Google Scholar 

Download references

Acknowledgements

Technical assistance by Frauke Grohmann, Dagmar Leisner and Iris Bauer is acknowledged. The project was funded by the German Research Society DFG (SCHA 677/9-1), the Sander Foundation (2010.076.1) and the German Cluster of Excellence ‘Inflammation-at-Interfaces’.

Author information

Author notes

  1. A Arlt and S Sebens: These authors contributed equally to this work.This work is part of a doctoral thesis (SK).

Authors and Affiliations

  1. Department of Internal Medicine I, Laboratory of Molecular Gastroenterology & Hepatology, UKSH, Kiel, Germany

    A Arlt, S Krebs, C Geismann, M Grossmann, M-L Kruse, S Schreiber & H Schäfer

  2. Institute of Experimental Medicine, Inflammatory Carcinogenesis Research Group, Kiel, Germany

    S Sebens

  3. Institute of Clinical Molecular Biology, UKSH, Kiel, Germany

    S Schreiber

Authors
  1. A Arlt
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. S Sebens
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. S Krebs
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. C Geismann
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. M Grossmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. M-L Kruse
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. S Schreiber
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. H Schäfer
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to H Schäfer.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Arlt, A., Sebens, S., Krebs, S. et al. Inhibition of the Nrf2 transcription factor by the alkaloid trigonelline renders pancreatic cancer cells more susceptible to apoptosis through decreased proteasomal gene expression and proteasome activity. Oncogene 32, 4825–4835 (2013). https://doi.org/10.1038/onc.2012.493

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2012.493

Keywords

This article is cited by

Search

Advanced search

Quick links