Annals of Surgical Oncology

, Volume 16, Issue 2, pp 534–543 | Cite as

Sulforaphane Stimulates Activation of Proapoptotic Protein Bax Leading to Apoptosis of Endothelial Progenitor Cells

  • Takeshi Nishikawa
  • Nelson H. Tsuno
  • Takeshi Tsuchiya
  • Satomi Yoneyama
  • Jun Yamada
  • Yasutaka Shuno
  • Yurai Okaji
  • Junichiro Tanaka
  • Joji Kitayama
  • Koki Takahashi
  • Hirokazu Nagawa
Laboratory and Translational Research


Sulforaphane (SUL) is an isothiocyanate naturally present in widely consumed vegetables, particularly in broccoli. SUL has recently been focused as a result of its inhibitory effects on tumor cell growth in vitro and in vivo. We used endothelial progenitor cells (EPCs) as an in vitro model to investigate the effect of SUL on the various steps of vasculogenesis and angiogenesis. Peripheral blood mononuclear cells from blood of normal human volunteers were plated on fibronectin-coated 100 mm dishes and incubated for 7 days. The viability of EPCs, treated with SUL at different doses, was assessed by MTS assay. Cell apoptosis was analyzed by flow cytometry. To determine the relative contributions of caspase-8 and caspase-9 pathways to SUL-induced apoptosis, the effect of caspase inhibitors was determined. The expression of apoptosis-related proteins (Bax, Bcl-2) was investigated by Western blot test. Finally, the effect of SUL on the ability of EPCs to form vascular-like structures on Matrigel was investigated. We clearly demonstrated that SUL induced the dose-dependent inhibition of EPCs’ viability by induction of apoptosis. All caspases (caspase-3, −8, and −9) were activated during apoptosis induction by SUL, but the effect of caspase-9 was more prominent than that of caspase-8. Also, the expression of Bax was upregulated by SUL treatment. In addition to apoptosis induction, SUL dose-dependently inhibited the tube-like formation by EPCs on Matrigel. The present results demonstrate the antivasculogenic/antiangiogenic activity of SUL in vitro and open premise for the use of SUL as a multipotent anticancer agent that targets both cancer cells and the angiogenic endothelium.


Endothelial Progenitor Cell Sulforaphane Cruciferous Vegetable Phenazine Methosulfate Chloromethyl Ketone 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Folkman J, Shing Y. Angiogenesis. J Biol Chem. 1992;267:10931–4.PubMedGoogle Scholar
  2. 2.
    Risau W, Sariola H, Zerwes HG, et al. Vasculogenesis and angiogenesis in embryonic-stem-cell-derived embryoid bodies. Development. 1988;102:471–8.PubMedGoogle Scholar
  3. 3.
    Okaji Y, Tsuno NH, Saito S, et al. Vaccines targeting tumour angiogenesis—a novel strategy for cancer immunotherapy. Eur J Surg Oncol. 2006;32:1–8.CrossRefGoogle Scholar
  4. 4.
    Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature. 2000;407:249–57.PubMedCrossRefGoogle Scholar
  5. 5.
    Asahara T, Murohara T, Sullivan, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997;275:964–7.Google Scholar
  6. 6.
    Asahara T, Takahashi T, Masuda H, et al. VEGF contributes to postnatal neovascularization by mobilizing bone marrow–derived endothelial progenitor cells. EMBO J. 1999;18:3964–72.PubMedCrossRefGoogle Scholar
  7. 7.
    Kalka C, Masuda H, Takahashi T, et al. Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc Natl Acad Sci USA. 2000;97:3422–7.PubMedCrossRefGoogle Scholar
  8. 8.
    Peters BA, Diaz LA Jr, Polyak K, et al. Contribution of bone marrow-derived endothelial cells to human tumor vasculature. Nat Med. 2005;11:261–2.PubMedCrossRefGoogle Scholar
  9. 9.
    Hutter R, Carrick FE, Valdiviezo C, et al. Vascular endothelial growth factor regulates reendothelialzation and neointima formation in a mouse model of arterial injury. Circulation. 2004;110:2430–5.PubMedCrossRefGoogle Scholar
  10. 10.
    Werner N, Kosiol S, Schiegl T, et al. Circulating endothelial progenitor cells and cardiovascular outcomes. N Engl J Med. 2005;353:999–1007.PubMedCrossRefGoogle Scholar
  11. 11.
    Rehman J, Li J, Orschell CM, et al. Peripheral blood “endothelial progenitor cells” are derived from monocyte/macrophages and secrete angiogenic growth factors. Circulation. 2003;107:1164–9.PubMedCrossRefGoogle Scholar
  12. 12.
    Asakage M, Tsuno NH, Kitayama J, et al. Early-outgrowth of endothelial progenitor cells can function as antigen-presenting cells. Cancer Immunol Immumother. 2006;55:708–16.CrossRefGoogle Scholar
  13. 13.
    Kohlmeier L, Mendez M. Controversies surrounding diet and breast cancer. Proc Nutr Soc. 1997;56:369–82.PubMedCrossRefGoogle Scholar
  14. 14.
    Zhang SM, Hunter DJ, Rosner BA, et al. Intakes of fruits, vegetables, and related nutrients and the risk of non-Hodgkin’s lymphoma among women. Cancer Epidemiol Biomarkers Prev. 2000;9:477–85.PubMedGoogle Scholar
  15. 15.
    Cohen JH, Kristal AR, Stanford JL. Fruit and vegetable intakes and prostate cancer risk. J Natl Cancer Inst. 2000;92:61–8.PubMedCrossRefGoogle Scholar
  16. 16.
    Kolonel LN, Hankin JH, Whittemore AS, et al. Vegetables, fruits, legumes and prostate cancer: a multiethnic case-control study. Cancer Epidemiol Biomarkers Prev. 2000;9:795–804.PubMedGoogle Scholar
  17. 17.
    Hecht SS. Inhibition of carcinogenesis by isothiocyantes. Drug Metab Rev. 2000;32:395–411.PubMedCrossRefGoogle Scholar
  18. 18.
    Fahey JW, Zalcmann AT, Talalay P. The chemical diversity and distribution of glucosinolates and isothiocyanates among plants. Phytochemistry. 2001;56:5–51.PubMedCrossRefGoogle Scholar
  19. 19.
    Gao X, Dinkova-Kostova AT, Talalay P. Powerful and prolonged protection of human retinal pigment epithelial cells, keratinocytes, and mouse leukemia cells against oxidative damage: the indirect antioxidant effects of sulforaphane. Proc Natl Acad Sci USA. 2001;98:15221–6.PubMedCrossRefGoogle Scholar
  20. 20.
    Fahey JW, Zhang Y, Talalay P. Broccoli sprout: an exceptionally rich source of inducers of enzymes that protect against chemical carcinogen. Proc Natl Acad Sci USA. 1997;94:10367–72.PubMedCrossRefGoogle Scholar
  21. 21.
    Zhang Y, Talalay P, Cho CG, et al. A major inducer of anticarcinogenic protective enzymes from broccoli: isolation and elucidation of structure. Proc Natl Acad Sci USA. 1992;89:2399–403.PubMedCrossRefGoogle Scholar
  22. 22.
    Gamet-Payrastre L, Li P, Lumeau S, et al. Sulforaphane, a naturally occurring isothiocyanate, induces cell cycle arrest and apoptosis in HT29 human colon cancer cells. Cancer Res. 2000;60:1426–33.PubMedGoogle Scholar
  23. 23.
    Bonnesen C, Eggleston IM, Hayes JD. Dietary indoles and isothiocyanates that are genereted from cruciferous vegetables can both stimulate apoptosis and confer protection against DNA damage in human colon cell lines. Cancer Res. 2001;61:6120–30.PubMedGoogle Scholar
  24. 24.
    Pappa G, Bartsch H, Gerhause C. Biphasic modulation of cell proliferation by sulforaphane at physiologically relevant exposure times in a human colon cancer cell line. Mol Nutr Food Res. 2007;51:977–84.PubMedCrossRefGoogle Scholar
  25. 25.
    Singh SV, Herman-Antosiewicz A, Singh AV, et al. Sulforaphane-induced G2/M phase cell cycle arrest involves checkpoint kinase 2-mediated phosphorylation of cell division cycle 25C. J Biol Chem. 2004;279:25813–22.PubMedCrossRefGoogle Scholar
  26. 26.
    Chiao JW, Chung FL, Kancherla R, et al. Sulforaphane and its metabolite mediate growth arrest and apoptosis in human prostate cancer cells. Int J Oncol. 2002;20:631–6.PubMedGoogle Scholar
  27. 27.
    Shan Y, Sun C, Zhao X, et al. Effect of sulforaphane on cell growth, G(0)/G(1) phase cell progression and apoptosis in human bladder cancer T24 cells. Int J Oncol. 2006;29:883–8.PubMedGoogle Scholar
  28. 28.
    Fimognari C, Nusse M, Berti F, et al. Cyclin D3 and p53 mediate sulforaphane-induced cell cycle delay and apoptosis in non-transformed human T lymphocytes. Cell Mol Life Sci. 2002;59:2004–12.PubMedCrossRefGoogle Scholar
  29. 29.
    Asakage M, Tsuno NH, Kitayama J, et al. Sulforaphane induces inhibition of human umbilical vein endothelial cells proliferation by apoptosis. Angiogenesis. 2006;9:83–91.PubMedCrossRefGoogle Scholar
  30. 30.
    Vasa M, Fichtlscherer S, Adler K, et al. Increase in circulating endothelial progenitor cells by statin therapy in patients with stable coronary artery disease. Circulation. 2001;103:2885–90.PubMedCrossRefGoogle Scholar
  31. 31.
    Hecht SS. Chemoprevention of cancer by isothiocyanates, modifiers of carcinogen metabolism. J Nutr. 1999;129:768S–74S.PubMedGoogle Scholar
  32. 32.
    Talalay P, Zhang Y. Chemoprotection against cancer by isothiocyanates and glucosinolates. Biochem Soc Trans. 1996;24:806–10.PubMedGoogle Scholar
  33. 33.
    Bertl E, Bartsch H, Gerhauser C. Inhibition of angiogenesis and endothelial cell functions are novel sulforaphane-mediated mechanisms in chemoprevention. Mol Cancer Ther. 2006;5:575–85.PubMedCrossRefGoogle Scholar
  34. 34.
    Green DR, Reed JC. Mitochondria and apoptosis. Science. 1998;281:1309–12.PubMedCrossRefGoogle Scholar
  35. 35.
    Wolter KG, Hsu YT, Smith CL, et al. Movement of bax from the cytosol to mitochondria during apoptosis. J Cell Biol. 1997;139:1281–92.PubMedCrossRefGoogle Scholar
  36. 36.
    Pastorino JG, Chen ST, Tafani M, et al. The overexpression of bax produces cell death upon induction of the mitochondrial permeability transition. J Biol Chem. 1998;273:7770–5.PubMedCrossRefGoogle Scholar
  37. 37.
    Narita M, Shimizu S, Ito T, et al. Bax interacts with permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria. Proc Natl Acad Sci USA. 1998;95:14681–6.PubMedCrossRefGoogle Scholar
  38. 38.
    Granville DJ, Carthy CM, Hunt DW, et al. Apoptosis: molecular aspects of cell death and disease. Lab Invest. 1998;78:893–913.PubMedGoogle Scholar
  39. 39.
    Yang YM, Conaway CC, Chiao JW, et al. Inhibition of benzo(a)pyrene-induced lung tumorigenesis in A/J mice by dietary N-acetylcysteine conjugates of benzyl and phenethyl isotiocyanates during the postinitiation phase is associated with activation of mitogen-activated protein kinases and p53 activity and induction of apoptosis. Cancer Res. 2002;62:2–7.PubMedGoogle Scholar
  40. 40.
    Matsuda K, Yoshida K, Taya Y, et al. p53AIP1 regulates the mitochondrial apoptotic pathway. Cancer Res. 2002;62:2883–9.PubMedGoogle Scholar
  41. 41.
    Shimizu S, Narita M, Tsujimoto Y. Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC. Nature. 1999;399:483–7.PubMedCrossRefGoogle Scholar
  42. 42.
    Kluck RM, Bossy-Wetzel E, Green DR, et al. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science. 1997;275:1132–6.PubMedCrossRefGoogle Scholar
  43. 43.
    Shintani S, Murohara T, Ikeda H, et al. Mobilization of endothelial progenitor cells in patients with acute myocardial infarction. Circulation. 2001;103:2776–9.PubMedCrossRefGoogle Scholar
  44. 44.
    Takahashi T, Kalka C, Masuda H, et al. Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat Med. 1999;5:434–8.PubMedCrossRefGoogle Scholar
  45. 45.
    Ishida A, Ohya Y, Sakuda H, et al. Autologous peripheral blood mononuclear cell implantation for patients with peripheral arterial disease improve limb ischemia. Circ J. 2005;69:1260–5.PubMedCrossRefGoogle Scholar
  46. 46.
    Walter DH, Rittig K, Bahlmann FH, et al. Statin therapy accelerates reendothelialization: a novel effect involving mobilization and incorporation of bone marrow-derived endothelial progenitor cells. Circulation. 2002;105:3017–24.PubMedCrossRefGoogle Scholar
  47. 47.
    Murohara T, Asahara T, Silver M, et al. Nitric oxide synthase modulates angiogenesis in response to tissue ischemia. J Clin Invest. 1998;101:2567–78.PubMedCrossRefGoogle Scholar
  48. 48.
    Sata M. Molecular strategies to treat vascular diseases: circulating vascular progenitor cell as a potential target for prophylactic treatment of atherosclerosis. Circ J. 2003;67:983–91.PubMedCrossRefGoogle Scholar
  49. 49.
    Gill M, Dias S, Hattori K, et al. Vascular trauma induces rapid but transient mobilization of VEGFR2(+) AC133(+) endothelial precursor cells. Circ Res. 2001;88:167–74.PubMedGoogle Scholar
  50. 50.
    Kawamoto A, Gwon HC, Iwaguro H, et al. Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia. Circulation. 2001;103:634–7.PubMedGoogle Scholar
  51. 51.
    Rausher FM, Goldschmidt-Clermont PJ, Davis BH, et al. Aging, progenitor cell exhaustion, and atherosclerosis. Circulation. 2003;108:457–63.CrossRefGoogle Scholar
  52. 52.
    Vasa M, Fichtlscherer S, Aicher A, et al. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ Res. 2001;89:E1–7.PubMedCrossRefGoogle Scholar
  53. 53.
    Hill JM, Zalos G, Halcox JP, et al. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med. 2003;348:593–600.PubMedCrossRefGoogle Scholar
  54. 54.
    Choi JH, Kim KL, Huh W, et al. Decreased number and impaired angiogenic function of endothelial progenitor cells in patients with chronic renal failure. Arterioscler Thromb Vasc Biol. 2004;24:1246–52.PubMedCrossRefGoogle Scholar
  55. 55.
    Okaji Y, Tsuno NH, Kitayama J, et al. Vaccination with autologous endothelium inhibits angiogenesis and metastasis of colon cancer through autoimmunity. Cancer Sci. 2004;95:85–90.PubMedCrossRefGoogle Scholar
  56. 56.
    Tsuchiya T, Okaji Y, Tsuno NH, et al. Targeting Id1 and Id3 inhibits peritoneal metastasis of gastric cancer. Cancer Sci. 2005;96:784–90.PubMedCrossRefGoogle Scholar
  57. 57.
    Yoneyama S, Okaji Y, Tsuno NH, et al. A study of dendritic and endothelial cell interactions in colon cancer in a cell line and small mammal model. Eur J Surg Oncol. 2007;1–8.Google Scholar
  58. 58.
    Zhang Y, Kensler TW, Cho CG, et al. Anticarcinogenic activities of sulforaphane and structurally related synthetic norbornyl isothiocyanates. Proc Natl Acad Sci USA. 1994;91:3147–50.PubMedCrossRefGoogle Scholar
  59. 59.
    Zhang Y, Talalay P. Mechanism of differential potencies of isothiocyantes as inducers of anticarcinogenic Phase 2 enzymes. Cancer Res. 1998;58:4632–9.PubMedGoogle Scholar
  60. 60.
    Prestera T, Talalay P. Electrophile and antioxidant regulation of enzymes that detoxify carcinogens. Proc Natl Acad Sci USA. 1995;92:8965–9.PubMedCrossRefGoogle Scholar
  61. 61.
    Ye L, Zhang Y. Total intracellular accumulation levels of dietary isothiocyanates determine their activity in elevation of cellular glutathione and induction of phase2 detoxification enzymes. Carcinogenesis. 2001;22:1987–92.PubMedCrossRefGoogle Scholar
  62. 62.
    Wattenberg LW. Inhibitory effects of benzyl isothiocyanate administered shortly before diethlnitrosamine or benzo(a)pyrene on pulmonary and forestomach neoplasia in A/J mice. Carcinogenesis. 1987;8:1971–3.PubMedCrossRefGoogle Scholar
  63. 63.
    Jiao D, Yu MC, Hankin JH, et al. Total isothiocyanate contents in cooked vegetables frequently consumed in Singapore. J Agric Food Chem. 1998;46:1055–8.CrossRefGoogle Scholar
  64. 64.
    Ye L, Dinkova-Kostova AT, Wade KL, Zhang Y, Shapiro TA, Talalay P. Quantitative determination of dithiocarbamates in human plasma, serum, erythrocytes and urine: pharmacokinetics of broccoli sprout isothiocyanates in humans. Clin Chim Acta. 2002;316:43–53.PubMedCrossRefGoogle Scholar

Copyright information

© Society of Surgical Oncology 2008

Authors and Affiliations

  • Takeshi Nishikawa
    • 1
  • Nelson H. Tsuno
    • 1
    • 2
  • Takeshi Tsuchiya
    • 1
  • Satomi Yoneyama
    • 1
  • Jun Yamada
    • 1
  • Yasutaka Shuno
    • 1
  • Yurai Okaji
    • 2
  • Junichiro Tanaka
    • 1
  • Joji Kitayama
    • 1
  • Koki Takahashi
    • 2
  • Hirokazu Nagawa
    • 1
  1. 1.Department of Surgical OncologyUniversity of TokyoTokyoJapan
  2. 2.Faculty of Medicine, Department of Transfusion MedicineUniversity of TokyoTokyoJapan

Personalised recommendations