Investigational New Drugs

, Volume 30, Issue 3, pp 898–915 | Cite as

Inhibition of TGF-β signaling, vasculogenic mimicry and proinflammatory gene expression by isoxanthohumol

  • Annegret Serwe
  • Kristina Rudolph
  • Timm Anke
  • Gerhard Erkel


TGF-β is a multifunctional cytokine that regulates cell proliferation, differentiation, apoptosis and extracellular matrix production. Deregulation of TGF-β production or signaling has been associated with a variety of pathological processes such as cancer, metastasis, angiogenesis and fibrosis. Therefore, TGF-β signaling has emerged as an attractive target for the development of new cancer therapeutics. In a screening program of natural compounds from fungi inhibiting the TGF-β dependent expression of a reporter gene in HepG2 cells, we found that the flavone isoxanthohumol inhibited the binding of the activated Smad2/3 transcription factors to the DNA and antagonized the cellular effects of TGF-β including reporter gene activation and expression of TGF-β induced genes in HepG2 and MDA-MB-231 cells. In an in vitro angiogenesis assay, isoxanthohumol (56 μM) strongly decreased the formation of capillary-like tubules of MDA-MB-231 cells on Matrigel. In addition, we found that isoxanthohumol blocked IFN-γ, IL-4 and IL-6 dependent Jak/Stat signaling and strongly inhibited the induction of pro-inflammatory genes in MonoMac6 cells at the transcriptional level after LPS/TPA treatment.


Isoxanthohumol TGF-β signaling Vasculogenic mimicry 



This work was supported by a grant from the Stiftung Rheinland-Pfalz für Innovation. We are very thankful to Prof. H. Anke for providing the crude extracts for the screening as well as Trichoderma harzianum IBWF278b-95. We thank Prof. S. Dooley, Medical Faculty of Mannheim, for providing the (AGCCAGACA)9MLP-Luc reporter plasmid, Prof. B. Brüne, University of Frankfurt, for providing the HepG2-pH3SVL cells, and Prof. O. Sterner, University of Lund, for the structure elucidation.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Siegel PM, Massague J (2003) Cytostatic and apoptotic actions of TGF-beta in homeostasis and cancer. Nat Rev Cancer 3:807–821PubMedCrossRefGoogle Scholar
  2. 2.
    Li MO, Wan YY, Sanjabi S, Robertson A-KL, Flavell RA (2006) Transforming growth factor-β regulation of immune responses. Annu Rev Immunol 24:99–149PubMedCrossRefGoogle Scholar
  3. 3.
    Massague J, Seoane J, Wotton D (2006) Smad transcription factors. Genes Dev 19:2783–2810CrossRefGoogle Scholar
  4. 4.
    Gordon KJ, Blobe GC (2008) Role of transforming growth factor-β super family signaling pathways in human disease. Biochim Biophys Acta 1782:197–228PubMedGoogle Scholar
  5. 5.
    Feng X-H, Derynck R (2005) Specificity and versatility in TGF-β signaling through Smads. Annu Rev Cell Dev Biol 21:659–693PubMedCrossRefGoogle Scholar
  6. 6.
    Moustakas A, Heldin CH (2005) Non-Smad TGF-β signals. J Cell Sci 118:3573–3584PubMedCrossRefGoogle Scholar
  7. 7.
    Pardali K, Moustakas A (2007) Actions of TGF-β as tumor suppressor and pro-metastatic factor in human cancer. Biochim Biophys Acta 1775:21–62PubMedGoogle Scholar
  8. 8.
    Bierie B, Moses HL (2006) TGFβ: the molecular Jekyll and Hyde of cancer. Nat Rev Cancer 6:506–520PubMedCrossRefGoogle Scholar
  9. 9.
    Leivonen S-K, Kähäri V-M (2007) Transforming growth factor-β signaling in cancer invasion and metastasis. Int J Cancer 121:2119–2124PubMedCrossRefGoogle Scholar
  10. 10.
    Bertolino P, Deckers M, Lebrin F, ten Dijke P (2005) Transforming growth factor-β signal transduction in angiogenesis and vascular disorders. Chest 128:585S–590SPubMedCrossRefGoogle Scholar
  11. 11.
    Wahl SM, Wen J, Moutsopoulos N (2006) TGF-β: a mobile purveyor of immune privilege. Immunol Rev 213:213–227PubMedCrossRefGoogle Scholar
  12. 12.
    Yingling JM, Blanchard KL, Sawyer S (2004) Development of TGF-β signaling inhibitors for cancer therapy. Nat Rev Drug Discov 3:1011–1022PubMedCrossRefGoogle Scholar
  13. 13.
    Iyer S, Wang Z-G, Akhtari M, Zhao W, Seth P (2005) Targeting TGFβ signaling for cancer therapy. Cancer Biol Ther 4:261–266PubMedCrossRefGoogle Scholar
  14. 14.
    Pinkas J, Teicher BA (2006) TGF-β in cancer and as therapeutic target. Biochem Pharmacol 72:523–529PubMedCrossRefGoogle Scholar
  15. 15.
    Mojzis J, Varinska L, Mojzisova G, Kostova I, Mirossay L (2008) Antiangiogenic effects of flavonoids and chalcones. Pharmacol Res 57:259–265PubMedCrossRefGoogle Scholar
  16. 16.
    Gerhäuser C (2005) Beer constituents as potential cancer chemo preventive agents. Eur J Cancer 41:1941–1954PubMedCrossRefGoogle Scholar
  17. 17.
    Juvvadi PR, Seshime Y, Kitamoto K (2005) Genomics reveals traces of fungal phenylpropanoid-flavonoid metabolic pathway in the filamentous fungus Aspergillus oryzae. J Microbiol 43:475–486PubMedGoogle Scholar
  18. 18.
    White TJ, Bruns T, Lee S, Taylor AW (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic Press, Inc, San Diego, pp 315–322Google Scholar
  19. 19.
    Stevens JF, Taylor AW, Deinzer ML (1999) Quantitative analysis of xanthohumol and related prenylflavonoids in hops and beer by liquid chromatography—tandem mass spectrometry. J Chromatogr A 832:97–107PubMedCrossRefGoogle Scholar
  20. 20.
    Roehm NW, Rodgers H, Hatfield SM, Glasebrook AL (1991) An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT. J Immunol Meth 142:257–265CrossRefGoogle Scholar
  21. 21.
    Erkel G, Belahmer H, Serwe A, Anke T, Kunz H, Kolshorn H, Liermann J, Opatz T (2008) Oxacyclododecindione, a novel inhibitor of IL-4 signaling from Exserohilum rostratum. J Antibiot (Tokyo) 61:285–290CrossRefGoogle Scholar
  22. 22.
    Dennler S, Itoh S, Vivien D, ten Dijke P, Huet S, Gauthier JM (1998) Direct binding of Smad3 and Smad4 to critical TGF beta-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene. EMBO J 17:3091–3100PubMedCrossRefGoogle Scholar
  23. 23.
    Mikita T, Campbell D, Wu P, Williamson K, Schindler U (1996) Requirements for interleukin-4-induced gene expression and functional characterization of STAT6. Mol Cell Biol 16:5811–5820PubMedGoogle Scholar
  24. 24.
    Weidler M, Rether J, Anke T, Erkel G (2000) Inhibition of interleukin-6 signaling by galiellalactone. FEBS Lett 484:1–6PubMedCrossRefGoogle Scholar
  25. 25.
    Rether J, Erkel G, Anke T, Sterner O (2004) Inhibition of inducible TNF-α expression by oxaspirodion, a novel spiro-compound from the ascomycete Chaetomium subspirale. Biol Chem 385:829–834PubMedCrossRefGoogle Scholar
  26. 26.
    Spurrell JCL, Wiehler S, Zaheer RS, Sanders SP, Proud D (2005) Human airway epithelial cells produce IP-10 (CXCL10) in vitro and in vivo upon rhinovirus infection. Am J Physiol Lung Cell Mol Physiol 289:85–95CrossRefGoogle Scholar
  27. 27.
    Ray S, Sherman CT, Lu M, Brasier AR (2002) Angiotensinogen gene expression is dependent on signal transducer and activator of transcription 3-mediated p300/cAMP response element binding protein-binding protein coactivator recruitment and histone acyltransferase activity. Mol Endocrinol 16:824–836PubMedCrossRefGoogle Scholar
  28. 28.
    Pahl HL, Baeuerle PA (1995) A novel signal transduction pathway from the endoplasmatic reticulum to the nucleus is mediated by transcription factor NF-κB. EMBO J 14:2580–2588PubMedGoogle Scholar
  29. 29.
    Lokker NA, Sullivan CM, Hollenbach SJ, Israel MA, Giese NA (2002) Platelet-derived growth factor (PDGF) autocrine signaling regulates survival and mitogenic pathways in glioblastoma cells: evidence that the novel PDGF-C and PDGF-D ligands may play a role in the development of brain tumors. Cancer Res 62:3729–3735PubMedGoogle Scholar
  30. 30.
    Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:2002–2007CrossRefGoogle Scholar
  31. 31.
    Liao D, Johnson RS (2007) Hypoxia: a key regulator of angiogenesis in cancer. Cancer Metastasis Rev 26:281–290PubMedCrossRefGoogle Scholar
  32. 32.
    Semenza GL (2010) Defining the role of hypoxia-inducible factor 1 in cancer biology and therapeutics. Oncogene 29:625–634PubMedCrossRefGoogle Scholar
  33. 33.
    Melillo G (2007) Targeting hypoxia cell signaling for cancer therapy. Cancer Metastasis Rev 26:341–352PubMedCrossRefGoogle Scholar
  34. 34.
    Abdollah S, Macias-Silva M, Tsukazaki T, Hayashi H, Attisano L, Wrana JL (1997) TßRI phosphorylation of Smad2 on Ser465 and Ser467 is required for Smad2-Smad4 complex formation and signaling. J Biol Chem 272:27678–27685PubMedCrossRefGoogle Scholar
  35. 35.
    Souchelnytskyi S, Tamaki K, Engstrom U, Wernstedt C, ten Dijke P, Heldin CH (1997) Phosphorylation of Ser465 and Ser467 in the C terminus of Smad2 mediates interaction with Smad4 and is required for transforming growth factor-beta signaling. J Biol Chem 272:28107–28115PubMedCrossRefGoogle Scholar
  36. 36.
    Liu X, Sun Y, Constantinescu SN, Karam E, Weinberg RA, Lodish HF (1997) Transforming growth factor beta-induced phosphorylation of Smad3 is required for growth inhibition and transcriptional induction in epithelial cells. Proc Natl Acad Sci USA 94:10669–10674PubMedCrossRefGoogle Scholar
  37. 37.
    Chen CR, Kang Y, Massague J (2001) Defective repression of c-myc in breast cancer cells: a loss at the core of the transforming growth factor β growth arrest program. Proc Natl Acad Sci USA 98:992–999PubMedCrossRefGoogle Scholar
  38. 38.
    Deng J, Grande F, Neamati N (2007) Small molecule inhibitors of Stat3 signaling pathway. Curr Cancer Drug Targets 7:91–107PubMedCrossRefGoogle Scholar
  39. 39.
    Klampfer L (2006) Signal transducers and activators of transcription (STATs): novel targets of chemo preventive and chemotherapeutic drugs. Curr Cancer Drug Targets 6:107–121PubMedCrossRefGoogle Scholar
  40. 40.
    Janknecht R, Wells NJ, Hunter T (1998) TGF-β-stimulated cooperation of Smad proteins with the coactivators CBP/p300. Genes Dev 12:2114–2119PubMedCrossRefGoogle Scholar
  41. 41.
    Simonsson M, Kanduri M, Grönroos E, Heldin C-H, Ericsson J (2006) The DNA binding activities of Smad2 and Smad3 are regulated by coactivator-mediated acetylation. J Biol Chem 281:39870–39880PubMedCrossRefGoogle Scholar
  42. 42.
    Hebenstreit D, Wirnsberger G, Horejs-Hoeck J, Duschl A (2006) Signaling mechanisms, interaction partners, and target genes of Stat6. Cytokine Growth Factor Rev 17:173–188PubMedCrossRefGoogle Scholar
  43. 43.
    Mantovani A, Allavena P, Sica A, Balkwill F (2008) Cancer-related inflammation. Nature 454:436–444PubMedCrossRefGoogle Scholar
  44. 44.
    Gaestel M, Kotlyarov A, Kracht M (2009) Targeting innate immunity protein kinase signalling in inflammation. Nat Rev Drug Discov 8:480–499PubMedCrossRefGoogle Scholar
  45. 45.
    Wrzesinski SH, Wan YY, Flavell RA (2007) Transforming growth factor-β and the immune response: Implications for anticancer therapy. Clin Cancer Res 13:5262–5270PubMedCrossRefGoogle Scholar
  46. 46.
    von Gersdorff G, Susztak K, Rezvani F, Bitzer M, Liang D, Boettinger EP (2000) Smad3 and Smad4 mediate transcriptional activation of the human Smad7 promoter by transforming growth factor β. J Biol Chem 275:11320–11326CrossRefGoogle Scholar
  47. 47.
    Javelaud D, Mauviel A (2005) Crosstalk mechanisms between the mitogen-activated protein kinase pathways and Smad signaling downstream of TGF-β: implications for carcinogenesis. Oncogene 24:5742–5750PubMedCrossRefGoogle Scholar
  48. 48.
    Zhang S, Zhang D, Sun B (2007) Vasculogenic mimicry: current status and future prospects. Cancer Lett 254:157–164PubMedCrossRefGoogle Scholar
  49. 49.
    Paulis YWJ, Soetekouw PMMB, Verheul HMW, Tjan-Heijnen VCG, Griffioen AW (2010) Signalling pathways in vasculogenic mimicry. Biochim Biophys Acta 1806:18–28PubMedGoogle Scholar
  50. 50.
    Reich NC, Liu L (2006) Tracking Stat nuclear traffic. Nat Rev Immunol 6:602–612PubMedCrossRefGoogle Scholar
  51. 51.
    Decker T, Kovarik P (2000) Serine phosphorylation of STATs. Oncogene 19:2628–2637PubMedCrossRefGoogle Scholar
  52. 52.
    Inoue Y, Itoh Y, Abe K, Okamoto T, Daitoku H, Fukamizu A, Onozaki K, Hayashi H (2007) Smad3 is acetylated by p300/CBP to regulate its transactivation activity. Oncogene 26:500–508PubMedCrossRefGoogle Scholar
  53. 53.
    Shankaranarayanan P, Chaitidis P, Kühn H, Nigam S (2001) Acetylation by histone acetyltransferase CREB-binding protein/p300 of Stat6 is required for transcriptional activation of the 15-lipoxygenase-1 gene. J Biol Chem 276:42753–42760PubMedCrossRefGoogle Scholar
  54. 54.
    Yu H, Jove R (2004) The Stats of cancer-new molecular targets come of age. Nat Rev Cancer 4:97–105PubMedCrossRefGoogle Scholar
  55. 55.
    Devarajan E, Huang S (2009) STAT3 as a central regulator of tumor metastases. Curr Mol Med 9:626–633PubMedCrossRefGoogle Scholar
  56. 56.
    Chen Z, Han ZC (2008) Stat3: a critical transcription activator in angiogenesis. Med Res Rev 28:185–200PubMedCrossRefGoogle Scholar
  57. 57.
    Berg T (2008) Signal transducers and activators of transcription as targets for small organic molecules. Chembiochem 9:2039–2044PubMedCrossRefGoogle Scholar
  58. 58.
    Lin W-W, Karin M (2007) A cytokine-mediated link between innate immunity, inflammation and cancer. J Clin Invest 117:1175–1183PubMedCrossRefGoogle Scholar
  59. 59.
    Aggarwal BB, Shishodia S, Sandur SK, Pandey MK, Sethi G (2006) Inflammation and cancer: how hot is the link? Biochem Pharmacol 72:1605–1621PubMedCrossRefGoogle Scholar
  60. 60.
    Nakanishi C, Toi M (2005) Nuclear factor-κB inhibitors as sensitizers to anticancer drugs. Nat Rev Cancer 5:297–309PubMedCrossRefGoogle Scholar
  61. 61.
    Gerhauser C, Alt A, Heiss E, Gamal-Eldeen A, Klimo K, Knauft J, Neumann I, Scherf H-R, Frank N, Bartsch H, Becker H (2002) Cancer chemo preventive activity of xanthohumol, a natural product derived from hop. Mol Cancer Ther 1:959–969PubMedGoogle Scholar
  62. 62.
    Albini A, Dell’Eva R, Vene R, Ferrari N, Buhler DR, Noonan DM, Fassina G (2006) Mechanisms of the antiangiogenic activity by the hop flavonoid xanthohumol: NF-kappaB and Akt as targets. FASEB J 20:527–529PubMedGoogle Scholar
  63. 63.
    Harikumar KB, Kunnumakkara AB, Ahn KS, Anand P, Krishnan S, Guha S, Aggarwal BB (2009) Modification of the cysteine residues in IκBα kinase and NF-κB (p65) by xanthohumol leads to suppression of NF-κB-regulated gene products and potentiation of apoptosis in leukemia cells. Blood 113:2003–2013PubMedCrossRefGoogle Scholar
  64. 64.
    Vanhoecke B, Derycke L, Van Marck V, Depypere H, De Keukeleire D, Bracke M (2005) Antiinvasive effect of xanthohumol, a prenylated chalcone present in hops (Humulus lupulus L.) and beer. Int J Cancer 117:889–895PubMedCrossRefGoogle Scholar
  65. 65.
    Monteiro R, Calhau C, e Silva AO, Pinheiro-Silva S, Guerreiro S, Gärtner F, Azevedo I, Soares R (2008) Xanthohumol Inhibits inflammatory factor production and angiogenesis in breast cancer xenografts. J Cell Biochem 04:1699–1707CrossRefGoogle Scholar
  66. 66.
    Cho Y-C, Kim HJ, Kim Y-J, Lee KY, Choi HJ, Lee I-S, Kang BY (2008) Differential anti-inflammatory pathway by xanthohumol in IFN-γ and LPS-activated macrophages. Int Immunopharmacol 8:567–573PubMedCrossRefGoogle Scholar
  67. 67.
    Bertl E, Becker H, Eicher T, Herhaus C, Kapadia G, Bartsch H, Gerhäuser C (2004) Inhibition of endothelial cell functions by novel potential cancer chemo preventive agents. Biochem Biophys Res Commun 325:287–295PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Annegret Serwe
    • 1
  • Kristina Rudolph
    • 1
  • Timm Anke
    • 2
  • Gerhard Erkel
    • 2
  1. 1.Institute of Biotechnology and Drug Research (IBWF e. V.)KaiserslauternGermany
  2. 2.Department of BiotechnologyUniversity of KaiserslauternKaiserslauternGermany

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