Molecular and Cellular Biochemistry

, Volume 337, Issue 1–2, pp 201–212 | Cite as

FOXO transcription factors and VEGF neutralizing antibody enhance antiangiogenic effects of resveratrol

  • Rakesh K. Srivastava
  • Terry G. Unterman
  • Sharmila Shankar


Resveratrol (trans-3,5,4′-trihydroxystilbene), a compound found largely in the skins of red grapes and wines, possesses anti-cancer and anti-angiogenic properties and protects the cardiovascular system. However, the molecular mechanisms by which resveratrol inhibits angiogenesis are currently subjects of intense investigation. The purpose of this study was to examine whether FOXO transcription factors mediate anti-angiogenic effects of resveratrol, and whether vascular endothelial growth factor (VEGF) neutralizing antibody can enhance these effects of resveratrol. Inhibition of PI3 kinase (PI3K)/AKT and MEK/ERK pathways synergistically inhibited migration and capillary tube formation of Human Umbilical Vein Endothelial Cells (HUVECs) and further enhanced the anti-angiogenic effects of resveratrol. Inhibitors of AKT and MEK kinase synergistically inhibited cytoplasmic FOXO3a phosphorylation, which was accompanied by its nuclear translocation in HUVECs. Interestingly, inhibition of PI3K/AKT and MEK/ERK pathways synergistically induced FOXO transcriptional activity and inhibited cell migration and capillary tube formation. Antiangiogenic effects of resveratrol were enhanced by inhibitors of AKT and MEK. Phosphorylation-deficient mutants of FOXOs induced FOXO transcriptional activity, inhibited HUVEC cell migration, and capillary tube formation, and also enhanced antiangiogenic effects of resveratrol. Finally, VEGF neutralizing antibody enhanced the anti-proliferative and anti-angiogenic effects of resveratrol. In conclusion, regulation of FOXO transcription factors by resveratrol may play an important role in angiogenesis which is critical for cancer, diabetic retinopathy, rheumatoid arthritis, psoriasis, and cardiovascular disorders.


Angiogenesis FOXO Resveratrol Vascular endothelial growth factors (VEGF) 


  1. 1.
    Shankar S, Singh G, Srivastava RK (2007) Chemoprevention by resveratrol: molecular mechanisms and therapeutic potential. Front Biosci 12:4839–4854CrossRefPubMedGoogle Scholar
  2. 2.
    Sagar SM, Yance D, Wong RK (2006) Natural health products that inhibit angiogenesis: a potential source for investigational new agents to treat cancer-Part 1. Curr Oncol 13:14–26PubMedGoogle Scholar
  3. 3.
    Shankar S, Siddiqui I, Srivastava RK (2007) Molecular mechanisms of resveratrol (3, 4, 5-trihydroxy-trans-stilbene) and its interaction with TNF-related apoptosis inducing ligand (TRAIL) in androgen-insensitive prostate cancer cells. Mol Cell Biochem 304:273–285CrossRefPubMedGoogle Scholar
  4. 4.
    Galili N, Davis RJ, Fredericks WJ, Mukhopadhyay S, Rauscher FJ III, Emanuel BS, Rovera G, Barr FG (1993) Fusion of a fork head domain gene to PAX3 in the solid tumour alveolar rhabdomyosarcoma. Nat Genet 5:230–235CrossRefPubMedGoogle Scholar
  5. 5.
    Anderson MJ, Viars CS, Czekay S, Cavenee WK, Arden KC (1998) Cloning and characterization of three human forkhead genes that comprise an FKHR-like gene subfamily. Genomics 47:187–189CrossRefPubMedGoogle Scholar
  6. 6.
    Hillion J, Le Coniat M, Jonveaux P, Berger R, Bernard OA (1997) AF6q21, a novel partner of the MLL gene in t(6;11)(q21;q23), defines a forkhead transcriptional factor subfamily. Blood 90:3714–3719PubMedGoogle Scholar
  7. 7.
    Borkhardt A, Repp R, Haas OA, Leis T, Harbott J, Kreuder J, Hammermann J, Henn T, Lampert F (1997) Cloning and characterization of AFX, the gene that fuses to MLL in acute leukemias with a t(X;11)(q13;q23). Oncogene 14:195–202CrossRefPubMedGoogle Scholar
  8. 8.
    Van Der Heide LP, Hoekman MF, Smidt MP (2004) The ins and outs of FoxO shuttling: mechanisms of FoxO translocation and transcriptional regulation. Biochem J 380:297–309CrossRefGoogle Scholar
  9. 9.
    Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, Anderson MJ, Arden KC, Blenis J, Greenberg ME (1999) Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96:857–868CrossRefPubMedGoogle Scholar
  10. 10.
    Guo S, Rena G, Cichy S, He X, Cohen P, Unterman T (1999) Phosphorylation of serine 256 by protein kinase B disrupts transactivation by FKHR and mediates effects of insulin on insulin-like growth factor-binding protein-1 promoter activity through a conserved insulin response sequence. J Biol Chem 274:17184–17192CrossRefPubMedGoogle Scholar
  11. 11.
    Medema RH, Kops GJ, Bos JL, Burgering BM (2000) AFX-like Forkhead transcription factors mediate cell-cycle regulation by Ras and PKB through p27kip1. Nature 404:782–787CrossRefPubMedGoogle Scholar
  12. 12.
    Nakamura N, Ramaswamy S, Vazquez F, Signoretti S, Loda M, Sellers WR (2000) Forkhead transcription factors are critical effectors of cell death and cell cycle arrest downstream of PTEN. Mol Cell Biol 20:8969–8982CrossRefPubMedGoogle Scholar
  13. 13.
    Manning BD, Cantley LC (2007) AKT/PKB signaling: navigating downstream. Cell 129:1261–1274CrossRefPubMedGoogle Scholar
  14. 14.
    Dijkers PF, Medema RH, Lammers JW, Koenderman L, Coffer PJ (2000) Expression of the pro-apoptotic Bcl-2 family member Bim is regulated by the forkhead transcription factor FKHR-L1. Curr Biol 10:1201–1204CrossRefPubMedGoogle Scholar
  15. 15.
    Tang TT, Dowbenko D, Jackson A, Toney L, Lewin DA, Dent AL, Lasky LA (2002) The forkhead transcription factor AFX activates apoptosis by induction of the BCL-6 transcriptional repressor. J Biol Chem 277:14255–14265CrossRefPubMedGoogle Scholar
  16. 16.
    Dijkers PF, Medema RH, Pals C, Banerji L, Thomas NS, Lam EW, Burgering BM, Raaijmakers JA, Lammers JW, Koenderman L, Coffer PJ (2000) Forkhead transcription factor FKHR-L1 modulates cytokine-dependent transcriptional regulation of p27(KIP1). Mol Cell Biol 20:9138–9148CrossRefPubMedGoogle Scholar
  17. 17.
    Cappellini A, Tabellini G, Zweyer M, Bortul R, Tazzari PL, Billi AM, Fala F, Cocco L, Martelli AM (2003) The phosphoinositide 3-kinase/Akt pathway regulates cell cycle progression of HL60 human leukemia cells through cytoplasmic relocalization of the cyclin-dependent kinase inhibitor p27(Kip1) and control of cyclin D1 expression. Leukemia 17:2157–2167CrossRefPubMedGoogle Scholar
  18. 18.
    Burgering BM, Kops GJ (2002) Cell cycle and death control: long live Forkheads. Trends Biochem Sci 27:352–360CrossRefPubMedGoogle Scholar
  19. 19.
    Tran H, Brunet A, Grenier JM, Datta SR, Fornace AJ Jr, DiStefano PS, Chiang LW, Greenberg ME (2002) DNA repair pathway stimulated by the forkhead transcription factor FOXO3a through the Gadd45 protein. Science 296:530–534CrossRefPubMedGoogle Scholar
  20. 20.
    Schmidt M, Fernandez de Mattos S, van der Horst A, Klompmaker R, Kops GJ, Lam EW, Burgering BM, Medema RH (2002) Cell cycle inhibition by FoxO forkhead transcription factors involves downregulation of cyclin D. Mol Cell Biol 22:7842–7852CrossRefPubMedGoogle Scholar
  21. 21.
    Maisonpierre PC, Suri C, Jones PF, Bartunkova S, Wiegand SJ, Radziejewski C, Compton D, McClain J, Aldrich TH, Papadopoulos N, Daly TJ, Davis S, Sato TN, Yancopoulos GD (1997) Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science 277:55–60CrossRefPubMedGoogle Scholar
  22. 22.
    Hosaka T, Biggs WH III, Tieu D, Boyer AD, Varki NM, Cavenee WK, Arden KC (2004) Disruption of forkhead transcription factor (FOXO) family members in mice reveals their functional diversification. Proc Natl Acad Sci USA 101:2975–2980CrossRefPubMedGoogle Scholar
  23. 23.
    Furuyama T, Kitayama K, Shimoda Y, Ogawa M, Sone K, Yoshida-Araki K, Hisatsune H, Nishikawa S, Nakayama K, Ikeda K, Motoyama N, Mori N (2004) Abnormal angiogenesis in Foxo1 (Fkhr)-deficient mice. J Biol Chem 279:34741–34749CrossRefPubMedGoogle Scholar
  24. 24.
    Daly C, Wong V, Burova E, Wei Y, Zabski S, Griffiths J, Lai KM, Lin HC, Ioffe E, Yancopoulos GD, Rudge JS (2004) Angiopoietin-1 modulates endothelial cell function and gene expression via the transcription factor FKHR (FOXO1). Genes Dev 18:1060–1071CrossRefPubMedGoogle Scholar
  25. 25.
    Yang B, Cao DJ, Sainz I, Colman RW, Guo YL (2004) Different roles of ERK and p38 MAP kinases during tube formation from endothelial cells cultured in 3-dimensional collagen matrices. J Cell Physiol 200:360–369CrossRefPubMedGoogle Scholar
  26. 26.
    Woessmann W, Meng YH, Mivechi NF (1999) An essential role for mitogen-activated protein kinases, ERKs, in preventing heat-induced cell death. J Cell Biochem 74:648–662CrossRefPubMedGoogle Scholar
  27. 27.
    Kyriakis JM, Avruch J (1996) Sounding the alarm: protein kinase cascades activated by stress and inflammation. J Biol Chem 271:24313–24316CrossRefPubMedGoogle Scholar
  28. 28.
    Asada S, Daitoku H, Matsuzaki H, Saito T, Sudo T, Mukai H, Iwashita S, Kako K, Kishi T, Kasuya Y, Fukamizu A (2007) Mitogen-activated protein kinases, Erk and p38, phosphorylate and regulate Foxo1. Cell Signal 19:519–527CrossRefPubMedGoogle Scholar
  29. 29.
    Dai J, Rabie AB (2007) VEGF: an essential mediator of both angiogenesis and endochondral ossification. J Dent Res 86:937–950CrossRefPubMedGoogle Scholar
  30. 30.
    Nagy JA, Benjamin L, Zeng H, Dvorak AM, Dvorak HF (2008) Vascular permeability, vascular hyperpermeability and angiogenesis. Angiogenesis 11:109–119CrossRefPubMedGoogle Scholar
  31. 31.
    Khosravi Shahi P, Fernandez Pineda I (2008) Tumoral angiogenesis: review of the literature. Cancer Invest 26:104–108CrossRefPubMedGoogle Scholar
  32. 32.
    Sirohi B, Smith K (2008) Bevacizumab in the treatment of breast cancer. Expert Rev Anticancer Ther 8:1559–1568CrossRefPubMedGoogle Scholar
  33. 33.
    Socinski MA (2008) Bevacizumab as first-line treatment for advanced non-small cell lung cancer. Drugs Today (Barc) 44:293–301CrossRefGoogle Scholar
  34. 34.
    Lien S, Lowman HB (2008) Therapeutic anti-VEGF antibodies. Handb Exp Pharmacol 181:131–150CrossRefPubMedGoogle Scholar
  35. 35.
    Beatty GL, Giantonio BJ (2008) Bevacizumab and oxaliplatin-based chemotherapy in metastatic colorectal cancer. Expert Rev Anticancer Ther 8:683–688CrossRefPubMedGoogle Scholar
  36. 36.
    Wheatley-Price P, Shepherd FA (2008) Targeting angiogenesis in the treatment of lung cancer. J Thorac Oncol 3:1173–1184CrossRefPubMedGoogle Scholar
  37. 37.
    Bradley DP, Tessier JJ, Lacey T, Scott M, Jurgensmeier JM, Odedra R, Mills J, Kilburn L, Wedge SR (2008) Examining the acute effects of cediranib (RECENTIN, AZD2171) treatment in tumor models: a dynamic contrast-enhanced MRI study using gadopentate. Magn Reson Imaging 27:377–384CrossRefPubMedGoogle Scholar
  38. 38.
    Heckman CA, Holopainen T, Wirzenius M, Keskitalo S, Jeltsch M, Yla-Herttuala S, Wedge SR, Jurgensmeier JM, Alitalo K (2008) The tyrosine kinase inhibitor cediranib blocks ligand-induced vascular endothelial growth factor receptor-3 activity and lymphangiogenesis. Cancer Res 68:4754–4762CrossRefPubMedGoogle Scholar
  39. 39.
    Lang SA, Brecht I, Moser C, Obed A, Batt D, Schlitt HJ, Geissler EK, Stoeltzing O (2008) Dual inhibition of Raf and VEGFR2 reduces growth and vascularization of hepatocellular carcinoma in an experimental model. Langenbecks Arch Surg 393:333–341CrossRefPubMedGoogle Scholar
  40. 40.
    Lang SA, Schachtschneider P, Moser C, Mori A, Hackl C, Gaumann A, Batt D, Schlitt HJ, Geissler EK, Stoeltzing O (2008) Dual targeting of Raf and VEGF receptor 2 reduces growth and metastasis of pancreatic cancer through direct effects on tumor cells, endothelial cells, and pericytes. Mol Cancer Ther 7:3509–3518CrossRefPubMedGoogle Scholar
  41. 41.
    Shankar S, Chen Q, Sarva K, Siddiqui I, Srivastava RK (2007) Curcumin enhances the apoptosis-inducing potential of TRAIL in prostate cancer cells: molecular mechanisms of apoptosis, migration and angiogenesis. J Mol Signal 2:10–18CrossRefPubMedGoogle Scholar
  42. 42.
    Furukawa-Hibi Y, Kobayashi Y, Chen C, Motoyama N (2005) FOXO transcription factors in cell-cycle regulation and the response to oxidative stress. Antioxid Redox Signal 7:752–760CrossRefPubMedGoogle Scholar
  43. 43.
    Shankar S, Chen Q, Srivastava RK (2008) Inhibition of PI3K/AKT and MEK/ERK pathways act synergistically to enhance antiangiogenic effects of EGCG through activation of FOXO transcription factor. J Mol Signal 3:7–16CrossRefPubMedGoogle Scholar
  44. 44.
    Kau TR, Schroeder F, Ramaswamy S, Wojciechowski CL, Zhao JJ, Roberts TM, Clardy J, Sellers WR, Silver PA (2003) A chemical genetic screen identifies inhibitors of regulated nuclear export of a Forkhead transcription factor in PTEN-deficient tumor cells. Cancer Cell 4:463–476CrossRefPubMedGoogle Scholar
  45. 45.
    Shankar S, Ganapathy S, Hingorani SR, Srivastava RK (2008) EGCG inhibits growth, invasion, angiogenesis and metastasis of pancreatic cancer. Front Biosci 13:440–452CrossRefPubMedGoogle Scholar
  46. 46.
    Chlench S, Mecha Disassa N, Hohberg M, Hoffmann C, Pohlkamp T, Beyer G, Bongrazio M, Da Silva-Azevedo L, Baum O, Pries AR, Zakrzewicz A (2007) Regulation of Foxo-1 and the angiopoietin-2/Tie2 system by shear stress. FEBS Lett 581:673–680CrossRefPubMedGoogle Scholar
  47. 47.
    Potente M, Urbich C, Sasaki K, Hofmann WK, Heeschen C, Aicher A, Kollipara R, DePinho RA, Zeiher AM, Dimmeler S (2005) Involvement of Foxo transcription factors in angiogenesis and postnatal neovascularization. J Clin Invest 115:2382–2392CrossRefPubMedGoogle Scholar
  48. 48.
    Daitoku H, Hatta M, Matsuzaki H, Aratani S, Ohshima T, Miyagishi M, Nakajima T, Fukamizu A (2004) Silent information regulator 2 potentiates Foxo1-mediated transcription through its deacetylase activity. Proc Natl Acad Sci USA 101:10042–10047CrossRefPubMedGoogle Scholar
  49. 49.
    Folkman J (2002) Role of angiogenesis in tumor growth and metastasis. Semin Oncol 29:15–18PubMedGoogle Scholar
  50. 50.
    Ellis LM, Hicklin DJ (2008) VEGF-targeted therapy: mechanisms of anti-tumour activity. Nat Rev Cancer 8:579–591CrossRefPubMedGoogle Scholar
  51. 51.
    Folkman J (2003) Fundamental concepts of the angiogenic process. Curr Mol Med 3:643–651CrossRefPubMedGoogle Scholar
  52. 52.
    Folkman J (2003) Angiogenesis and proteins of the hemostatic system. J Thromb Haemost 1:1681–1682CrossRefPubMedGoogle Scholar
  53. 53.
    Folkman J (2003) Angiogenesis inhibitors: a new class of drugs. Cancer Biol Ther 2:S127–S133PubMedGoogle Scholar
  54. 54.
    Folkman J (2003) Angiogenesis and apoptosis. Semin Cancer Biol 13:159–167CrossRefPubMedGoogle Scholar
  55. 55.
    Tang TT, Lasky LA (2003) The forkhead transcription factor FOXO4 induces the down-regulation of hypoxia-inducible factor 1 alpha by a von Hippel-Lindau protein-independent mechanism. J Biol Chem 278:30125–30135CrossRefPubMedGoogle Scholar
  56. 56.
    Huang H, Tindall DJ (2006) FOXO factors: a matter of life and death. Future Oncol 2:83–89CrossRefPubMedGoogle Scholar
  57. 57.
    Potente M, Fisslthaler B, Busse R, Fleming I (2003) 11, 12-Epoxyeicosatrienoic acid-induced inhibition of FOXO factors promotes endothelial proliferation by down-regulating p27Kip1. J Biol Chem 278:29619–29625CrossRefPubMedGoogle Scholar
  58. 58.
    Folkman J (2007) Angiogenesis: an organizing principle for drug discovery? Nat Rev Drug Discov 6:273–286CrossRefPubMedGoogle Scholar
  59. 59.
    Kerbel RS (2008) Tumor angiogenesis. N Engl J Med 358:2039–2049CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2009

Authors and Affiliations

  • Rakesh K. Srivastava
    • 1
    • 2
  • Terry G. Unterman
    • 3
    • 4
  • Sharmila Shankar
    • 5
  1. 1.Department of Pharmacology, Toxicology and TherapeuticsThe University of Kansas Medical CenterKansas CityUSA
  2. 2.Department of MedicineThe University of Kansas Medical CenterKansas CityUSA
  3. 3.Department of Medicine, College of Medicine and Jesse Brown VA Medical CenterUniversity of Illinois at ChicagoChicagoUSA
  4. 4.Department of Physiology and Biophysics, College of Medicine and Jesse Brown VA Medical CenterUniversity of Illinois at ChicagoChicagoUSA
  5. 5.Department of Pathology & Laboratory MedicineThe University of Kansas Medical CenterKansas CityUSA

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