Molecular Biology Reports

, Volume 39, Issue 5, pp 5529–5535 | Cite as

Apigenin enhances the cytotoxic effects of tumor necrosis factor-related apoptosis-inducing ligand in human rheumatoid arthritis fibroblast-like synoviocytes

  • Qing-wen Sun
  • Song-min Jiang
  • Ke Yang
  • Jian-ming Zheng
  • Li Zhang
  • Wei-dong Xu


Activated rheumatoid arthritis (RA) fibroblast-like synoviocytes (RAFLSs) play a central role in both initiating and driving RA. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been documented to induce apoptosis only in a small proportion of RAFLSs, which is followed by an induction of proliferation in surviving cells. Apigenin, a chemopreventive bioflavonoid, exhibits proapoptotic activity in many types of cells. In the present study, we sought to determine whether apigenin could enhance the cytotoxic effect of TRAIL on activated RAFLSs. Human RAFLSs isolated from patients with RA were treated with TRAIL (1 nM), apigenin (20 μM), or their combination, and subjected to apoptosis analysis after a 24-h incubation and proliferation analysis after a 72-h incubation. Apoptosis assay revealed that TRAIL or apigenin alone induced a marked apoptosis in RAFLS and their combination yielded a synergistic increase in RAFLS apoptosis. Immunoblotting analysis of apoptosis regulators demonstrated that combined treatment with apigenin increased caspase-3 expression and activity and decreased the Bcl-2/Bax ratio relative to treatment with TRAIL alone. The presence of apigenin significantly restrained TRAIL-induced RAFLS proliferation, coupled with restoration of the expression of two cell-cycle inhibitors p21 and p27. Moreover, the combination with apigenin blunted TRAIL-induced activation of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. Our data collectively demonstrate that apigenin sensitizes RAFLS to TRAIL-induced apoptosis and counteracts TRAIL-dependent RAFLS proliferation, which is likely mediated through inactivation of PI3-K/Akt signaling pathway.


Rheumatoid arthritis Fibroblast-like synoviocytes Apigenin Tumor necrosis factor-related apoptosis inducing ligand Akt Apoptosis Proliferation 



This work was supported by the National Natural Science Foundation of China (31070747).


  1. 1.
    Firestein GS (1996) Invasive fibroblast-like synoviocytes in rheumatoid arthritis: passive responders or transformed aggressors? Arthritis Rheum 39:1781–1790PubMedCrossRefGoogle Scholar
  2. 2.
    Cooles FA, Isaacs JD (2011) Pathophysiology of rheumatoid arthritis. Curr Opin Rheumatol 23:233–240PubMedCrossRefGoogle Scholar
  3. 3.
    Lafyatis R, Remmers EF, Roberts AB, Yocum DE, Sporn MB, Wilder RL (1989) Anchorage-independent growth of synoviocytes from arthritic and normal joints. Stimulation by exogenous platelet-derived growth factor and inhibition by transforming growth factor-beta and retinoids. J Clin Invest 83:1267–1276PubMedCrossRefGoogle Scholar
  4. 4.
    Müller-Ladner U, Kriegsmann J, Franklin BN, Matsumoto S, Geiler T, Gay RE, Gay S (1996) Synovial fibroblasts of patients with rheumatoid arthritis attach to and invade normal human cartilage when engrafted into SCID mice. Am J Pathol 149:1607–1615PubMedGoogle Scholar
  5. 5.
    Pap T, Müller-Ladner U, Gay RE, Gay S (2000) Fibroblast biology. Role of synovial fibroblasts in the pathogenesis of rheumatoid arthritis. Arthritis Res 2:361–367PubMedCrossRefGoogle Scholar
  6. 6.
    Pattacini L, Boiardi L, Casali B, Salvarani C (2010) Differential effects of anti-TNF-alpha drugs on fibroblast-like synoviocyte apoptosis. Rheumatology (Oxford) 49:480–489CrossRefGoogle Scholar
  7. 7.
    Pope RM (2002) Apoptosis as a therapeutic tool in rheumatoid arthritis. Nat Rev Immunol 2:527–535PubMedCrossRefGoogle Scholar
  8. 8.
    Baier A, Meineckel I, Gay S, Pap T (2003) Apoptosis in rheumatoid arthritis. Curr Opin Rheumatol 15:274–279PubMedCrossRefGoogle Scholar
  9. 9.
    Korb A, Pavenstädt H, Pap T (2009) Cell death in rheumatoid arthritis. Apoptosis 14:447–454PubMedCrossRefGoogle Scholar
  10. 10.
    Palao G, Santiago B, Galindo M, Pallá M, Ramírez JC, Pablos JL (2004) Downregulation of FLIP sensitizes rheumatoid synovial fibroblasts to Fas-mediated apoptosis. Arthritis Rheum 50:2803–2810PubMedCrossRefGoogle Scholar
  11. 11.
    Franz JK, Pap T, Hummel KM, Nawrath M, Aicher WK, Shigeyama Y, Müller-Ladner U, Gay RE, Gay S (2000) Expression of sentrin, a novel antiapoptotic molecule, at sites of synovial invasion in rheumatoid arthritis. Arthritis Rheum 43:599–607PubMedCrossRefGoogle Scholar
  12. 12.
    Perlman H, Georganas C, Pagliari LJ, Koch AE, Haines KIII, Pope RM (2000) Bcl-2 expression in synovial fibroblasts is essential for maintaining mitochondrial homeostasis and cell viability. J Immunol 164:5227–5235PubMedGoogle Scholar
  13. 13.
    Liu H, Eksarko P, Temkin V, Haines GK III, Perlman H, Koch AE, Thimmapaya B, Pope RM (2005) Mcl-1 is essential for the survival of synovial fibroblasts in rheumatoid arthritis. J Immunol 175:8337–8345PubMedGoogle Scholar
  14. 14.
    Miyashita T, Kawakami A, Tamai M, Izumi Y, Mingguo H, Tanaka F, Abiru S, Nakashima K, Iwanaga N, Aratake K, Kamachi M, Arima K, Ida H, Migita K, Origuchi T, Tagashira S, Nishikaku F, Eguchi K (2003) Akt is an endogenous inhibitor toward tumor necrosis factor-related apoptosis inducing ligand-mediated apoptosis in rheumatoid synovial cells. Biochem Biophys Res Commun 312:397–404PubMedCrossRefGoogle Scholar
  15. 15.
    Tsokos GC, Tsokos M (2003) The TRAIL to arthritis. J Clin Invest 112:1315–1317PubMedGoogle Scholar
  16. 16.
    Ichikawa K, Liu W, Fleck M, Zhang H, Zhao L, Ohtsuka T, Wang Z, Liu D, Mountz JD, Ohtsuki M, Koopman WJ, Kimberly R, Zhou T (2003) TRAIL-R2 (DR5) mediates apoptosis of synovial fibroblasts in rheumatoid arthritis. J Immunol 171:1061–1069PubMedGoogle Scholar
  17. 17.
    Perlman H, Nguyen N, Liu H, Eslick J, Esser S, Walsh K, Moore TL, Pope RM (2003) Rheumatoid arthritis synovial fluid macrophages express decreased tumor necrosis factor-related apoptosis-inducing ligand R2 and increased decoy receptor tumor necrosis factor-related apoptosis-inducing ligand R3. Arthritis Rheum 48:3096–3101PubMedCrossRefGoogle Scholar
  18. 18.
    Morel J, Audo R, Hahne M, Combe B (2005) Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces rheumatoid arthritis synovial fibroblast proliferation through mitogen-activated protein kinases and phosphatidylinositol 3-kinase/Akt. J Biol Chem 280:15709–15718PubMedCrossRefGoogle Scholar
  19. 19.
    Ross JA, Kasum CM (2002) Dietary flavonoids: bioavailability, metabolic effects, and safety. Annu Rev Nutr 22:19–34PubMedCrossRefGoogle Scholar
  20. 20.
    Clere N, Faure S, Martinez MC, Andriantsitohaina R (2011) Anticancer properties of flavonoids: roles in various stages of carcinogenesis. Cardiovasc Hematol Agents Med Chem 9:62–77PubMedCrossRefGoogle Scholar
  21. 21.
    Lu HF, Chie YJ, Yang MS, Lu KW, Fu JJ, Yang JS, Chen HY, Hsia TC, Ma CY, Ip SW, Chung JG (2010) Apigenin induces apoptosis in human lung cancer H460 cells through caspase- and mitochondria-dependent pathways. Hum Exp Toxicol 30(8):1053–1061. doi: 10.1177/0960327110386258 PubMedCrossRefGoogle Scholar
  22. 22.
    Kim BR, Jeon YK, Nam MJ (2011) A mechanism of apigenin-induced apoptosis is potentially related to anti-angiogenesis and anti-migration in human hepatocellular carcinoma cells. Food Chem Toxicol 49:1626–1632PubMedCrossRefGoogle Scholar
  23. 23.
    Bruno A, Siena L, Gerbino S, Ferraro M, Chanez P, Giammanco M, Gjomarkaj M, Pace E (2011) Apigenin affects leptin/leptin receptor pathway and induces cell apoptosis in lung adenocarcinoma cell line. Eur J Cancer 47(13):2042–2051. doi: 10.1016/j.ejca.2011.03.034 PubMedCrossRefGoogle Scholar
  24. 24.
    Horinaka M, Yoshida T, Shiraishi T, Nakata S, Wakada M, Sakai T (2006) The dietary flavonoid apigenin sensitizes malignant tumor cells to tumor necrosis factor-related apoptosis-inducing ligand. Mol Cancer Ther 5:945–951PubMedCrossRefGoogle Scholar
  25. 25.
    Arnett FC, Edworthy SM, Bloch DA et al (1988) The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 31:315–324PubMedCrossRefGoogle Scholar
  26. 26.
    Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63PubMedCrossRefGoogle Scholar
  27. 27.
    Audo R, Combe B, Coulet B, Morel J, Hahne M (2009) The pleiotropic effect of TRAIL on tumor-like synovial fibroblasts from rheumatoid arthritis patients is mediated by caspases. Cell Death Differ 16:1227–1237PubMedCrossRefGoogle Scholar
  28. 28.
    Shin GC, Kim C, Lee JM, Cho WS, Lee SG, Jeong M, Cho J, Lee K (2009) Apigenin-induced apoptosis is mediated by reactive oxygen species and activation of ERK1/2 in rheumatoid fibroblast-like synoviocytes. Chem Biol Interact 182:29–36PubMedCrossRefGoogle Scholar
  29. 29.
    Lee JH, Zhou HY, Cho SY, Kim YS, Lee YS, Jeong CS (2007) Anti-inflammatory mechanisms of apigenin: inhibition of cyclooxygenase-2 expression, adhesion of monocytes to human umbilical vein endothelial cells, and expression of cellular adhesion molecules. Arch Pharm Res 30:1318–1327PubMedCrossRefGoogle Scholar
  30. 30.
    Porter AG, Jänicke RU (1999) Emerging roles of caspase-3 in apoptosis. Cell Death Differ 6:99–104PubMedCrossRefGoogle Scholar
  31. 31.
    Adams JM, Cory S (2001) Life-or-death decisions by the Bcl-2 protein family. Trends Biochem Sci 26:61–66PubMedCrossRefGoogle Scholar
  32. 32.
    Kluck RM, Bossy-Wetzel E, Green DR, Newmeyer DD (1997) The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 275:1132–1136PubMedCrossRefGoogle Scholar
  33. 33.
    Torkin R, Lavoie JF, Kaplan DR, Yeger H (2005) Induction of caspase-dependent, p53-mediated apoptosis by apigenin in human neuroblastoma. Mol Cancer Ther 4:1–11PubMedGoogle Scholar
  34. 34.
    Levina V, Marrangoni AM, Demarco R, Gorelik E, Lokshin AE (2008) Multiple effects of TRAIL in human carcinoma cells: induction of apoptosis, senescence, proliferation, and cytokine production. Exp Cell Res 314:1605–1616PubMedCrossRefGoogle Scholar
  35. 35.
    Abukhdeir AM, Park BH (2008) P21 and p27: roles in carcinogenesis and drug resistance. Expert Rev Mol Med 10:e19PubMedCrossRefGoogle Scholar
  36. 36.
    Coqueret O (2003) New roles for p21 and p27 cell-cycle inhibitors: a function for each cell compartment? Trends Cell Biol 13:65–70PubMedCrossRefGoogle Scholar
  37. 37.
    Choi EJ, Kim GH (2009) Apigenin causes G(2)/M arrest associated with the modulation of p21(Cip1) and Cdc2 and activates p53-dependent apoptosis pathway in human breast cancer SK-BR-3 cells. J Nutr Biochem 20:285–290PubMedCrossRefGoogle Scholar
  38. 38.
    Trochon V, Blot E, Cymbalista F, Engelmann C, Tang RP, Thomaïdis A, Vasse M, Soria J, Lu H, Soria C (2000) Apigenin inhibits endothelial-cell proliferation in G(2)/M phase whereas it stimulates smooth-muscle cells by inhibiting P21 and P27 expression. Int J Cancer 85:691–696PubMedCrossRefGoogle Scholar
  39. 39.
    Hussain AR, Khan AS, Ahmed SO, Ahmed M, Platanias LC, Al-Kuraya KS, Uddin S (2010) Apigenin induces apoptosis via downregulation of S-phase kinase-associated protein 2-mediated induction of p27Kip1 in primary effusion lymphoma cells. Cell Prolif 43:170–183PubMedCrossRefGoogle Scholar
  40. 40.
    Kelly-Spratt KS, Philipp-Staheli J, Gurley KE, Hoon-Kim K, Knoblaugh S, Kemp CJ (2009) Inhibition of PI-3 K restores nuclear p27Kip1 expression in a mouse model of Kras-driven lung cancer. Oncogene 28:3652–3662PubMedCrossRefGoogle Scholar
  41. 41.
    Banerji L, Glassford J, Lea NC, Thomas NS, Klaus GG, Lam EW (2001) BCR signals target p27(Kip1) and cyclin D2 via the PI3-K signalling pathway to mediate cell cycle arrest and apoptosis of WEHI 231 B cells. Oncogene 20:7352–7367PubMedCrossRefGoogle Scholar
  42. 42.
    Cretney E, Shanker A, Yagita H, Smyth MJ, Sayers TJ (2006) TNF-related apoptosis-inducing ligand as a therapeutic agent in autoimmunity and cancer. Immunol Cell Biol 84:87–98PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  1. 1.Central Laboratory of Shanghai Chest Hospital Affiliated to Shanghai Jiaotong University Medical CollegeShanghaiChina
  2. 2.State Key Laboratory of Genetic Engineering, School of Life SciencesFudan UniversityShanghaiChina
  3. 3.Department of PathologyShanghai Changhai Hospital Affiliated to Second Military UniversityShanghaiChina
  4. 4.Department of OrthopedicsShanghai Changhai Hospital Affiliated to Second Military UniversityShanghaiChina

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