Abstract
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.
Similar content being viewed by others
References
Shankar S, Singh G, Srivastava RK (2007) Chemoprevention by resveratrol: molecular mechanisms and therapeutic potential. Front Biosci 12:4839–4854
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 13:14–26
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 304:273–285
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 5:230–235
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 47:187–189
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 90:3714–3719
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 14:195–202
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 380:297–309
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 96:857–868
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 274:17184–17192
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 404:782–787
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 20:8969–8982
Manning BD, Cantley LC (2007) AKT/PKB signaling: navigating downstream. Cell 129:1261–1274
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 10:1201–1204
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 277:14255–14265
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 20:9138–9148
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 17:2157–2167
Burgering BM, Kops GJ (2002) Cell cycle and death control: long live Forkheads. Trends Biochem Sci 27:352–360
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 296:530–534
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 22:7842–7852
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 277:55–60
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 101:2975–2980
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 279:34741–34749
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 18:1060–1071
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 200:360–369
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 74:648–662
Kyriakis JM, Avruch J (1996) Sounding the alarm: protein kinase cascades activated by stress and inflammation. J Biol Chem 271:24313–24316
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 19:519–527
Dai J, Rabie AB (2007) VEGF: an essential mediator of both angiogenesis and endochondral ossification. J Dent Res 86:937–950
Nagy JA, Benjamin L, Zeng H, Dvorak AM, Dvorak HF (2008) Vascular permeability, vascular hyperpermeability and angiogenesis. Angiogenesis 11:109–119
Khosravi Shahi P, Fernandez Pineda I (2008) Tumoral angiogenesis: review of the literature. Cancer Invest 26:104–108
Sirohi B, Smith K (2008) Bevacizumab in the treatment of breast cancer. Expert Rev Anticancer Ther 8:1559–1568
Socinski MA (2008) Bevacizumab as first-line treatment for advanced non-small cell lung cancer. Drugs Today (Barc) 44:293–301
Lien S, Lowman HB (2008) Therapeutic anti-VEGF antibodies. Handb Exp Pharmacol 181:131–150
Beatty GL, Giantonio BJ (2008) Bevacizumab and oxaliplatin-based chemotherapy in metastatic colorectal cancer. Expert Rev Anticancer Ther 8:683–688
Wheatley-Price P, Shepherd FA (2008) Targeting angiogenesis in the treatment of lung cancer. J Thorac Oncol 3:1173–1184
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 27:377–384
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 68:4754–4762
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 393:333–341
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 7:3509–3518
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 2:10–18
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 7:752–760
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 3:7–16
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 4:463–476
Shankar S, Ganapathy S, Hingorani SR, Srivastava RK (2008) EGCG inhibits growth, invasion, angiogenesis and metastasis of pancreatic cancer. Front Biosci 13:440–452
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 581:673–680
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 115:2382–2392
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 101:10042–10047
Folkman J (2002) Role of angiogenesis in tumor growth and metastasis. Semin Oncol 29:15–18
Ellis LM, Hicklin DJ (2008) VEGF-targeted therapy: mechanisms of anti-tumour activity. Nat Rev Cancer 8:579–591
Folkman J (2003) Fundamental concepts of the angiogenic process. Curr Mol Med 3:643–651
Folkman J (2003) Angiogenesis and proteins of the hemostatic system. J Thromb Haemost 1:1681–1682
Folkman J (2003) Angiogenesis inhibitors: a new class of drugs. Cancer Biol Ther 2:S127–S133
Folkman J (2003) Angiogenesis and apoptosis. Semin Cancer Biol 13:159–167
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 278:30125–30135
Huang H, Tindall DJ (2006) FOXO factors: a matter of life and death. Future Oncol 2:83–89
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 278:29619–29625
Folkman J (2007) Angiogenesis: an organizing principle for drug discovery? Nat Rev Drug Discov 6:273–286
Kerbel RS (2008) Tumor angiogenesis. N Engl J Med 358:2039–2049
Acknowledgments
We thank our lab members for critical reading of the manuscript. We also thank Dr. Noboru Motoyama (National Institute for Longevity Sciences, Obu, Aichi, Japan) and Dr. Tatsuo Furuyama (Sonoda Women’s University, Amagasaki, Hyogo, Japan) for providing FOXO expression plasmids and FOXO-luciferase construct (pGL3-6X DBE), respectively. The study was initiated at the University of Texas Health Science Center at Tyler. The project was supported by NIH R01CA114469 (R.K.S.) and the Department of Veterans Affairs Merit Review Program (T.G.U.).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Srivastava, R.K., Unterman, T.G. & Shankar, S. FOXO transcription factors and VEGF neutralizing antibody enhance antiangiogenic effects of resveratrol. Mol Cell Biochem 337, 201–212 (2010). https://doi.org/10.1007/s11010-009-0300-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11010-009-0300-5