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 Paper
  • Published:

Independent actions on cyclin-dependent kinases and aryl hydrocarbon receptor mediate the antiproliferative effects of indirubins

Oncogene volume 23, pages 4400–4412 (2004)Cite this article

Abstract

Indirubin, a bis-indole obtained from various natural sources, is responsible for the reported antileukemia activity of a Chinese Medicinal recipe, Danggui Longhui Wan. However, its molecular mechanism of action is still not well understood. In addition to inhibition of cyclin-dependent kinases and glycogen synthase kinase-3, indirubins have been reported to activate the aryl hydrocarbon receptor (AhR), a cotranscriptional factor. Here, we confirm the interaction of AhR and indirubin using a series of indirubin derivatives and show that their binding modes to AhR and to protein kinases are unrelated. As reported for other AhR ligands, binding of indirubins to AhR leads to its nuclear translocation. Furthermore, the apparent survival of AhR−/− and +/+ cells, as measured by the MTT assay, is equally sensitive to the kinase-inhibiting indirubins. Thus, the cytotoxic effects of indirubins are AhR-independent and more likely to be linked to protein kinase inhibition. In contrast, a dramatic cytostatic effect, as measured by actual cell counts and associated with a sharp G1 phase arrest, is induced by 1-methyl-indirubins, a subfamily of AhR-active but kinase-inactive indirubins. As shown for TCDD (dioxin), this effect appears to be mediated through the AhR-dependent expression of p27KIP1. Altogether these results suggest that AhR activation, rather than kinase inhibition, is responsible for the cytostatic effects of some indirubins. In contrast, kinase inhibition, rather than AhR activation, represents the main mechanism underlying the cytotoxic properties of this class of promising antitumor molecules.

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

AhR:

aryl hydrocarbon receptor

ARNT:

aryl hydrocarbon receptor nuclear translocator

bHLH/PAS:

basic helix–loop–helix/period-aryl hydrocarbon receptor nuclear translocator-single minded

BIO:

6-bromo-indirubin-3′-oxime

BSA:

bovine serum albumin

CDK:

cyclin-dependent kinase

DAPI:

diaminophenylindole, dihydrochloride

DTT:

dithiothreitol

EGFP:

enhanced green fluorescent protein

FCS:

fetal calf serum

FICZ:

6-formylindolo[3,2-b]carbazole

GSK-3:

glycogen synthase kinase -3

IO:

indirubin-3′-oxime

MeBIO:

1-methyl-6-bromo-indirubin-3′-oxime

MeIO:

1-methyl-indirubin-3′-oxime

MTT:

(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide

TCDD:

2,3,7,8-tetrachlorodibenzo-p-dioxin

XRE:

xenobiotic-responsive element

References

  • Adachi J, Mori Y, Matsui S, Takigami H, Fujino J, Kitagawa H, Miller III CA, Kato T, Saeki K and Matsuda T . (2001). J. Biol. Chem., 276, 31475–31478.

  • Andersson P, McGuire J, Rubio C, Gradin K, Whitelaw ML, Pettersson S, Hanberg A and Poellinger L . (2002). Proc. Natl. Acad. Sci. USA, 99, 9990–9995.

  • Abdelrahim M, Smith III R and Safe S . (2003). Mol. Pharmacol., 63, 1373–1381.

  • Bain J, McLauchlan H, Elliott M and Cohen P . (2003). Biochem. J., 371, 199–204.

  • Balfour-Paul J . (1998). Indigo. British Museum Press: London, UK, 264pp.

    Google Scholar 

  • Bergander L, Wahlstrom N, Alsberg T, Bergman J, Rannug A and Rannug U . (2003). Drug Metab. Dispos., 31, 233–241.

  • Borgne A and Meijer L . (1996). J. Biol. Chem., 271, 27847–27854.

  • Bradshaw TD, Trapani V, Vasselin DA and Westwell AD . (2002). Curr. Pharm. Design, 8, 2475–2490.

  • Chotani G, Dodge T, Hsu A, Kumar M, LaDuca R, Trimbur D, Weyler W and Sanford K . (2000). Biochim. Biophys. Acta, 1543, 434–455.

  • Cooksey CJ . (2001). Molecules, 6, 736–769.

  • Damiens E, Baratte B, Marie D, Eisenbrand G and Meijer L . (2001). Oncogene, 20, 3786–3797.

  • Davies TG, Tunnah P, Meijer L, Marko D, Eisenbrand G, Endicott JA and Noble MEM . (2001). Structure, 9, 389–397.

  • Dealler SF, Hawkey PM and Millar MR . (1988). J. Clin. Microbiol., 26, 2152–2156.

  • Denison MS, Phelen D and Elferink CJ . (1998). Xenobiotics, Receptors and Gene Expression. Denison MS and Helferich W (eds). Taylor & Francis: Philadelphia, PA, pp. 3–33.

  • Denison MS and Nagy SR . (2003). Annu. Rev. Pharmacol. Toxicol., 43, 309–334.

  • Elbi C, Misteli T and Hager GL . (2002). Mol. Biol. Cell, 13, 2001–2015.

  • Elferink CJ . (2003). Cell cycle regulators as therapeutic targets. Meijer L, Jezequel A and Roberge M (eds). Progr. Cell Cycle Res., 5, 261–267.

    Google Scholar 

  • Elferink CJ, Ge NL and Levine A . (2001). Mol. Pharmacol., 59, 664–673.

  • Fernandez-Salguero P, Pineau T, Hilbert DM, McPhail T, Lee SS, Kimura S, Nebert D, Rudikoff S, Ward JM and Gonzalez FJ . (1995). Science, 268, 722–726.

  • Ffrench M, Bryon PA, Fière D, Vu Van H, Gentilhomme O, Adeleine P and Viala J-J . (1985). Cytometry, 6, 47–53.

  • Fukunaga BN, Probst MR, Reisz-Porszasz S and Hankinson O . (1995). J. Biol. Chem., 270, 29270–29278.

  • Ge NL and Elferink CJ . (1998). J. Biol. Chem., 273, 22708–22713.

  • Gillam EMJ, Notley LM, Cai H, DeVoss JJ and Guengerich FP . (2000). Biochemistry, 39, 13817–13824.

  • Guthrie C and Fink GR . (1991). Guide to Yeast Genetics and Molecular Biology, Abelson JN and Simon MI (eds). Academic Press Inc.: San Diego, CA, USA.

    Google Scholar 

  • Hahn ME . (2002). Chem.-Biol. Interact., 141, 131–160.

  • Hankinson O . (1995). Annu. Rev. Pharmacol. Toxicol., 35, 307–340.

  • Hayashibara T, Yamada Y, Mori N, Harasawa H, Sugahara K, Miyanishi T, Kamihira S and Tomonaga M . (2003). Biochem. Biophys. Res. Commun., 300, 128–134.

  • Heath-Pagliuso Rogers WJ, Tullis K, Seidel SD, Cenijn PH, Brouwer A and Denison M . (1998). Biochemistry, 37, 11508–11515.

  • Hoessel R, Leclerc S, Endicott J, Noble M, Lawrie A, Tunnah P, Leost M, Damiens E, Marie D, Marko D, Niederberger E, Tang W, Eisenbrand G and Meijer L . (1999). Nat. Cell Biol., 1, 60–67.

  • Jacobs MN, Dickins M and Lewis DF . (2003). J. Steroid Biochem. Mol. Biol., 84, 117–132.

  • Jain S, Maltepe E, Lu MM, Simon C and Bradfield CA . (1998). Mech. Dev., 73, 117–123.

  • Joiakim A, Mathieu PA, Palermo C, Gasiewicz TA and Reiners JJ . (2003). Drug Metab. Dispos., 31, 1279–1282.

  • Kawanishi M, Sakamoto M, Ito A, Kishi K and Yagi T . (2003). Mutat. Res., 540, 99–105.

  • Knockaert M, Greengard P and Meijer L . (2002). Trends Pharmacol. Sci., 23, 417–425.

  • Koliopanos A, Kleeff J, Xiao Y, Safe S, Zimmermann A, Buchler MW and Friess H . (2002). Oncogene, 21, 6059–6070.

  • Kolluri SK, Weiss C, Koff A and Göttlicher M . (1999). Genes Dev., 13, 1742–1753.

  • Kolluri SK, Balduf C, Hofmann M and Göttlicher M . (2001). Cancer Res., 61, 8534–8539.

  • Kuchenhoff A, Seliger G, Klonisch T, Tscheudschilsuren G, Kaltwasser P, Seliger E, Buchmann J and Fischer B . (1999). Fertil. Steril., 71, 354–360.

  • Kuramoto N, Baba K, Gion K, Sugiyama C, Taniura H and Yoneda Y . (2003). J. Neurochem., 85, 264–273.

  • Lahvis GP, Lindell SL, Thomas RS, McCuskey RS, Murphy C, Glover E, Bentz M, Southard J and Bradfield CA . (2000). Proc. Natl. Acad. Sci. USA, 97, 10442–10447.

  • Leclerc S, Garnier M, Hoessel R, Marko D, Bibb JA, Snyder GL, Greengard P, Biernat J, Mandelkow E-M, Eisenbrand G and Meijer L . (2001). J. Biol. Chem., 276, 251–260.

  • Ma Q and Whitlock JP . (1996). Mol. Cell. Biol., 16, 2144–2150.

  • MacNeil IA, Tiong CL, Minor C, August PR, Grossman TH, Loiacono KA, Lynch BA, Phillips T, Narula S, Sundaramoorthi R, Tyler A, Aldredge T, Long H, Gilman M, Holt D and Osburne MS . (2001). J. Mol. Microbiol. Biotechnol., 3, 301–308.

  • Marko D, Schätzle S, Friedel A, Genzlinger A, Zankl H, Meijer L and Eisenbrand G . (2001). Br. J. Cancer, 84, 283–289.

  • Maugard T, Enaud E, Choisy P and Legoy MD . (2001). Phytochemistry, 58, 897–904.

  • Meijer L, Skaltsounis AL, Magiatis P, Polychronopoulos P, Knockaert M, Leost M, Ryan XP, Vonica CD, Brivanlou A, Dajani R, Tarricone A, Musacchio A, Roe SM, Pearl L and Greengard P . (2003). Chem. Biol., 10, 1255–1266.

  • Miller CA . (1997). J. Biol. Chem., 272, 32824–32829.

  • Miller CA . (1999). Toxicol. Appl. Pharmacol., 160, 297–303.

  • Mosmann J . (1983). Immunol. Methods, 65, 55–63.

  • Nagy SR, Sanborn JR, Hammock BD and Denison MS . (2002a). Toxicol. Sci., 65, 200–210.

  • Nagy SR, Liu G, Lam KS and Denison MS . (2002b). Biochemistry, 41, 861–868.

  • Peters WL and Wiley D . (1995). Toxicol. Appl. Pharmacol., 13, 214–221.

  • Petersen SL, Curran MA, Marconi SA, Carpenter CD, Lubbers LS and McAbee MD . (2000). J. Comp. Neurol., 427, 428–439.

  • Polychronopoulos P, Magiatis P, Skaltsounis L, Myrianthopoulos V, Mikros E, Tarricone A, Musacchio A, Roe SM, Pearl L, Leost M, Greengard P and Meijer L . (2004). J. Med. Chem., 47, 935–946.

  • Primot A, Baratte B, Gompel M, Borgne A, Liabeuf S, Romette JL, Costantini F and Meijer L . (2000). Protein Expr. Purif., 20, 394–404.

  • Procopio M, Lahm A, Tramontano A, Bonati L and Pitea D . (2002). Eur. J. Biochem., 269, 13–18.

  • Rannug U, Bramstedt H and Nilsson U . (1992). Mutat. Res., 282, 219–225.

  • Rowlands JG and Gustafsson JA . (1997). Crit. Rev. Toxicol., 27, 109–134.

  • Safe S . (2001). Toxicol. Lett., 120, 1–7.

  • Safe S and McDougal A . (2002). Int. J. Oncol., 20, 1123–1128.

  • Salomon-Nguyen F, Della-Valle V, Mauchauffe M, Busson-Le Coniat M, Ghysdael J, Berger R and Bernard OA . (2000). Proc. Natl. Acad. Sci. USA, 97, 6757–6762.

  • Santini RP, Myrand S, Elferink C and Reiners JJ . (2001). J. Pharmacol. Exp. Ther., 299, 718–728.

  • Schmidt JV, Su GH, Reddy JK, Simon MC and Bradfield CA . (1996). Proc. Natl. Acad. Sci. USA, 93, 6731–6736.

  • Shimizu Y, Nakatsuru Y, Ichinose M, Takahashi Y, Kume H, Mimura J, Fujii-Kuriyama Y and Ishikawa T . (2000). Proc. Natl. Acad. Sci. USA, 97, 779–782.

  • Song J, Clagett-Dame M, Peterson RE, Hahn ME, Westler WM, Sicinski RR and DeLuca HF . (2002). Proc. Natl. Acad. Sci. USA, 99, 14694–14699.

  • Spink BC, Hussain HM, Katz BH, Eisele L and Spink DC . (2003). Biochem. Pharmacol., 66, 2313–2321.

  • Tang W and Eisenbrand G . (1992). Chinese Drugs of Plant Origin, Chemistry, Pharmacology, and Use in Traditional and Modern Medicine. Springer-Verlag: Heidelberg.

    Book  Google Scholar 

  • Wei YD, Bergander L, Rannug U and Rannug A . (2000). Arch. Biochem. Biophys., 383, 99–107.

  • Weiss C, Kolluri SK, Kiefer F and Göttlicher M . (1996). Exp. Cell Res., 226, 154–163.

  • Wolff S, Harper PA, Wong JM, Mostert V, Wang Y and Abel J . (2001). Mol. Pharmacol., 59, 716–724.

  • Xiao Z, Hao Y, Liu B and Qian L . (2002). Leukemia Lymphoma, 43, 1763–1768.

  • Yoon BI, Hirabayashi Y, Kawasaki Y, Kodama Y, Kaneko T, Kanno J, Kim DY, Fujii-Kuriyama Y and Inoue T . (2002). Toxicol. Sci., 70, 150–156.

Download references

Acknowledgements

We are most thankful to our colleagues for providing reagents: Dr Gerhard Eisenbrand (compounds 3, 5, 6, 7, 8, 9, 19, 21, 22), Dr Martin Göttlicher (5L and BP8 cell lines), Dr Charles A Miller III (AhR agonists reporter yeast strain), and Dr Steve Safe (TCDD). This research was also supported by grants from the ‘Association pour la Recherche sur le Cancer’ ARC5732 (LM), ARC5812 (MB) and ARC5664 (MF), the National Institutes of Environmental Health Sciences NIH5P42ES04699 (MD) and by a grant (‘Molécules & Cibles Thérapeutiques’) from the ‘Ministère de la Recherche/INSERM/CNRS’ (LM).

Author information

Authors and Affiliations

  1. C.N.R.S., Cell Cycle Group & UPS-2682, Station Biologique, BP 74, 29682 ROSCOFF cedex, Bretagne, France

    Marie Knockaert, Marc Blondel, Stéphane Bach, Maryse Leost & Laurent Meijer

  2. Laboratory of Receptor Biology & Gene Expression, Bldg 41, Room B602, National Cancer Institute, NIH, Bethesda, 20892-5055, MD, USA

    Cem Elbi & Gordon L Hager

  3. Department of Environmental Toxicology, Meyer Hall, One Shields Avenue, University of California, Davis, 95616-8588, CA, USA

    Scott R Nagy, Dalho Han & Michael Denison

  4. Université Claude Bernard, Laboratoire de Cytologie Analytique et Cytogénétique Moléculaire, 8 Avenue Rockefeller, Lyon, 69373, Cedex 08, France

    Martine Ffrench

  5. Laboratory of Molecular & Cellular Neuroscience, The Rockefeller University, 1230 York Avenue, New York, 10021, NY, USA

    Xiaozhou P Ryan, Paul Greengard & Laurent Meijer

  6. Division of Pharmacognosy and Natural Products Chemistry, Department of Pharmacy, University of Athens, Panepistimiopolis Zografou, Athens, GR-15771, Greece

    Prokopios Magiatis, Panos Polychronopoulos & Leandros Skaltsounis

Authors
  1. Marie Knockaert
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Marc Blondel
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Stéphane Bach
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Maryse Leost
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Cem Elbi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Gordon L Hager
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Scott R Nagy
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Dalho Han
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Michael Denison
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Martine Ffrench
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Xiaozhou P Ryan
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Prokopios Magiatis
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Panos Polychronopoulos
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Paul Greengard
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Leandros Skaltsounis
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Laurent Meijer
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Laurent Meijer.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Knockaert, M., Blondel, M., Bach, S. et al. Independent actions on cyclin-dependent kinases and aryl hydrocarbon receptor mediate the antiproliferative effects of indirubins. Oncogene 23, 4400–4412 (2004). https://doi.org/10.1038/sj.onc.1207535

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.onc.1207535

Keywords

This article is cited by

Search

Advanced search

Quick links