Cancer Chemotherapy and Pharmacology

, Volume 69, Issue 1, pp 195–206 | Cite as

5, 7-Dimethoxyflavone sensitizes TRAIL-induced apoptosis through DR5 upregulation in hepatocellular carcinoma cells

Original Article



5, 7-dimethoxyflavone (DMF) has been reported to induce apoptosis in various cancer cells. The aim of this study was to examine whether DMF sensitizes human hepatocellular carcinoma (HCC) cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis and its mechanism.


Human hepatocellular carcinoma cell lines Hep3B, Huh-7, and Hep G2 and human embryo liver L-02 cells were cultured in vitro. The cytotoxic activities were determined using MTT assay. The apoptotic cell death was examined using Flow cytometry using PI staining and DNA agarose gel electrophoresis. The activities of caspase-3, caspase-8, and caspase-9 were measured using ELISA. Intracellular ROS was measured by FCM using the fluorescent probe DCHF-DA, and the expression of DR4, DR5, CHOP, GPR78, and ATF4 proteins was analyzed using Western blot.


Our results demonstrated subtoxic concentrations of DMF sensitize HCC cells to TRAIL-induced apoptosis and induce the death receptor 5 (DR5) expression level, accompanying the generation of reactive oxygen species (ROS) and the upregulation of CHOP, GPR78, and ATF4 protein expression. Pretreatment with N-acetylcysteine (NAC) inhibited DMF-induced upregulation of DR5, CHOP, GPR78, and ATF4 protein expression and blocked the cotreatment-induced apoptosis. Furthermore, DMF-mediated sensitization of HCC cells to TRAIL was reduced by administration of a blocking antibody or small interfering RNAs for DR5, salubrinal, an inhibitor of ER stress, and the small interfering RNAs for CHOP. However, DMF could not induce the upregulation of DR5 expression, generation of ROS, and sensitization of TRAIL-induced apoptotic cell death in human embryo liver L-02 cells or normal human peripheral blood mononuclear cells (PBMCs).


The present study demonstrates that DMF selectively enhances TRAIL-induced apoptosis by ROS-stimulated ER-stress triggering CHOP-mediated DR5 upregulation in HCC.


Hepatocellular carcinoma 5, 7-Dimethoxyflavone TRAIL DR5 Reactive oxygen species 



The work was supported by the Major Project of Scientific Research Fund of Hunan Province Education Department (NO. 09A054).


  1. 1.
    Wicker CA, Sahu RP, Kulkarni-Datar K, Srivastava SK, Brown TL (2010) BITC sensitizes pancreatic adenocarcinomas to TRAIL-induced apoptosis. Cancer Growth Metastasis 2009:45–55PubMedGoogle Scholar
  2. 2.
    Tiwary R, Yu W, Li J, Park SK, Sanders BG et al (2010) Role of endoplasmic reticulum stress in alpha-TEA mediated TRAIL/DR5 death receptor dependent apoptosis. PLoS One 5:e11865PubMedCrossRefGoogle Scholar
  3. 3.
    Sung B, Ravindran J, Prasad S, Pandey MK, Aggarwal BB (2010) Gossypol induces death receptor-5 through activation of ROS-ERK-chop pathway and sensitizes colon cancer cells to trail. J Biol Chem 285:35418–35427PubMedCrossRefGoogle Scholar
  4. 4.
    Seo SB, Hur JG, Kim MJ, Lee JW, Kim HB et al (2010) TRAIL sensitize MDR cells to MDR-related drugs by down-regulation of P-glycoprotein through inhibition of DNA-PKcs/Akt/GSK-3beta pathway and activation of caspases. Mol Cancer 9:199PubMedCrossRefGoogle Scholar
  5. 5.
    do Lim Y, Park JH (2009) Induction of p53 contributes to apoptosis of HCT-116 human colon cancer cells induced by the dietary compound fisetin. Am J Physiol Gastrointest Liver Physiol 296:G1060–G1068CrossRefGoogle Scholar
  6. 6.
    Poulaki V, Mitsiades CS, McMullan C, Fanourakis G, Negri J et al (2005) Human retinoblastoma cells are resistant to apoptosis induced by death receptors: role of caspase-8 gene silencing. Invest Ophthalmol Vis Sci 46:358–366PubMedCrossRefGoogle Scholar
  7. 7.
    Srivastava RK (2001) TRAIL/Apo-2L: mechanisms and clinical applications in cancer. Neoplasia 3:535–546PubMedCrossRefGoogle Scholar
  8. 8.
    Wang S, El-Deiry WS (2003) TRAIL and apoptosis induction by TNF-family death receptors. Oncogene 22:8628–8633PubMedCrossRefGoogle Scholar
  9. 9.
    Ivanov VN, Bhoumik A, Ronai Z (2003) Death receptors and melanoma resistance to apoptosis. Oncogene 22:3152–3161PubMedCrossRefGoogle Scholar
  10. 10.
    Ozoren N, Fisher MJ, Kim K, Liu CX, Genin A et al (2000) Homozygous deletion of the death receptor DR4 gene in a nasopharyngeal cancer cell line is associated with TRAIL resistance. Int J Oncol 16:917–925PubMedGoogle Scholar
  11. 11.
    Parkin DM (2001) Global cancer statistics in the year 2000. Lancet Oncol 2:533–543PubMedCrossRefGoogle Scholar
  12. 12.
    Carr BI (2004) Hepatocellular carcinoma: current management and future trends. Gastroenterology 127:S218–S224PubMedCrossRefGoogle Scholar
  13. 13.
    Shankar S, Srivastava RK (2004) Enhancement of therapeutic potential of TRAIL by cancer chemotherapy and irradiation: mechanisms and clinical implications. Drug Resist Updat 7:139–156PubMedCrossRefGoogle Scholar
  14. 14.
    Yamanaka T, Shiraki K, Sugimoto K, Ito T, Fujikawa K et al (2000) Chemotherapeutic agents augment TRAIL-induced apoptosis in human hepatocellular carcinoma cell lines. Hepatology 32:482–490PubMedCrossRefGoogle Scholar
  15. 15.
    Patanasethanont D, Nagai J, Yumoto R, Murakami T, Sutthanut K et al (2007) Effects of Kaempferia parviflora extracts and their flavone constituents on P-glycoprotein function. J Pharm Sci 96:223–233PubMedCrossRefGoogle Scholar
  16. 16.
    Wu D, Nair MG, DeWitt DL (2002) Novel compounds from Piper methysticum Forst (Kava Kava) roots and their effect on cyclooxygenase enzyme. J Agric Food Chem 50:701–705PubMedCrossRefGoogle Scholar
  17. 17.
    Haberlein H, Tschiersch KP, Schafer HL (1994) Flavonoids from Leptospermum scoparium with affinity to the benzodiazepine receptor characterized by structure activity relationships and in vivo studies of a plant extract. Pharmazie 49:912–922PubMedGoogle Scholar
  18. 18.
    Wen X, Walle T (2007) Cytochrome P450 1B1, a novel chemopreventive target for benzo[a]pyrene-initiated human esophageal cancer. Cancer Lett 246:109–114PubMedCrossRefGoogle Scholar
  19. 19.
    Wen X, Walle UK, Walle T (2005) 5, 7-Dimethoxyflavone downregulates CYP1A1 expression and benzo[a]pyrene-induced DNA binding in Hep G2 cells. Carcinogenesis 26:803–809PubMedCrossRefGoogle Scholar
  20. 20.
    Wen X, Walle T (2005) Preferential induction of CYP1B1 by benzo[a]pyrene in human oral epithelial cells: impact on DNA adduct formation and prevention by polyphenols. Carcinogenesis 26:1774–1781PubMedCrossRefGoogle Scholar
  21. 21.
    Wang CK, Cao JG, Peng B, Gu YX, Zheng GP et al (2010) Inhibition of growth and motility of human A549 lung carcinoma cells by a recombined vascular basement membrane derived peptide. Cancer Lett 292:261–268PubMedCrossRefGoogle Scholar
  22. 22.
    Yang XH, Zheng X, Cao JG, Xiang HL, Liu F et al (2010) 8-Bromo-7-methoxychrysin-induced apoptosis of hepatocellular carcinoma cells involves ROS and JNK. World J Gastroenterol 16:3385–3393PubMedCrossRefGoogle Scholar
  23. 23.
    Bodmer JL, Holler N, Reynard S, Vinciguerra P, Schneider P et al (2000) TRAIL receptor-2 signals apoptosis through FADD and caspase-8. Nat Cell Biol 2:241–243PubMedCrossRefGoogle Scholar
  24. 24.
    Sprick MR, Weigand MA, Rieser E, Rauch CT, Juo P et al (2000) FADD/MORT1 and caspase-8 are recruited to TRAIL receptors 1 and 2 and are essential for apoptosis mediated by TRAIL receptor 2. Immunity 12:599–609PubMedCrossRefGoogle Scholar
  25. 25.
    Kischkel FC, Lawrence DA, Chuntharapai A, Schow P, Kim KJ et al (2000) Apo2L/TRAIL-dependent recruitment of endogenous FADD and caspase-8 to death receptors 4 and 5. Immunity 12:611–620PubMedCrossRefGoogle Scholar
  26. 26.
    Benov L, Sztejnberg L, Fridovich I (1998) Critical evaluation of the use of hydroethidine as a measure of superoxide anion radical. Free Radic Biol Med 25:826–831PubMedCrossRefGoogle Scholar
  27. 27.
    LeBel CP, Ischiropoulos H, Bondy SC (1992) Evaluation of the probe 2′, 7′-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress. Chem Res Toxicol 5:227–231PubMedCrossRefGoogle Scholar
  28. 28.
    Pellerito O, Calvaruso G, Portanova P, De Blasio A, Santulli A et al (2010) The synthetic cannabinoid WIN 55, 212-2 sensitizes hepatocellular carcinoma cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by activating p8/CCAAT/enhancer binding protein homologous protein (CHOP)/death receptor 5 (DR5) axis. Mol Pharmacol 77:854–863PubMedCrossRefGoogle Scholar
  29. 29.
    Jo M, Kim TH, Seol DW, Esplen JE, Dorko K et al (2000) Apoptosis induced in normal human hepatocytes by tumor necrosis factor-related apoptosis-inducing ligand. Nat Med 6:564–567PubMedCrossRefGoogle Scholar
  30. 30.
    Leverkus M, Neumann M, Mengling T, Rauch CT, Brocker EB et al (2000) Regulation of tumor necrosis factor-related apoptosis-inducing ligand sensitivity in primary and transformed human keratinocytes. Cancer Res 60:553–559PubMedGoogle Scholar
  31. 31.
    Lawrence D, Shahrokh Z, Marsters S, Achilles K, Shih D et al (2001) Differential hepatocyte toxicity of recombinant Apo2L/TRAIL versions. Nat Med 7:383–385PubMedCrossRefGoogle Scholar
  32. 32.
    Qin J, Chaturvedi V, Bonish B, Nickoloff BJ (2001) Avoiding premature apoptosis of normal epidermal cells. Nat Med 7:385–386PubMedCrossRefGoogle Scholar
  33. 33.
    He Q, Huang Y, Sheikh MS (2004) Proteasome inhibitor MG132 upregulates death receptor 5 and cooperates with Apo2L/TRAIL to induce apoptosis in Bax-proficient and -deficient cells. Oncogene 23:2554–2558PubMedCrossRefGoogle Scholar
  34. 34.
    Woo JH, Kim YH, Choi YJ, Kim DG, Lee KS et al (2003) Molecular mechanisms of curcumin-induced cytotoxicity: induction of apoptosis through generation of reactive oxygen species, down-regulation of Bcl-XL and IAP, the release of cytochrome c and inhibition of Akt. Carcinogenesis 24:1199–1208PubMedCrossRefGoogle Scholar
  35. 35.
    Zou W, Liu X, Yue P, Zhou Z, Sporn MB et al (2004) c-Jun NH2-terminal kinase-mediated up-regulation of death receptor 5 contributes to induction of apoptosis by the novel synthetic triterpenoid methyl-2-cyano-3, 12-dioxooleana-1, 9-dien-28-oate in human lung cancer cells. Cancer Res 64:7570–7578PubMedCrossRefGoogle Scholar
  36. 36.
    Staib F, Hussain SP, Hofseth LJ, Wang XW, Harris CC (2003) TP53 and liver carcinogenesis. Hum Mutat 21:201–216PubMedCrossRefGoogle Scholar
  37. 37.
    Walle T, Ta N, Kawamori T, Wen X, Tsuji PA et al (2007) Cancer chemopreventive properties of orally bioavailable flavonoids-methylated versus unmethylated flavones. Biochem Pharmacol 73:1288–1296PubMedCrossRefGoogle Scholar
  38. 38.
    Kim EH, Yoon MJ, Kim SU, Kwon TK, Sohn S et al (2008) Arsenic trioxide sensitizes human glioma cells, but not normal astrocytes, to TRAIL-induced apoptosis via CCAAT/enhancer-binding protein homologous protein-dependent DR5 up-regulation. Cancer Res 68:266–275PubMedCrossRefGoogle Scholar
  39. 39.
    Lim JH, Park JW, Kim SH, Choi YH, Choi KS et al (2008) Rottlerin induces pro-apoptotic endoplasmic reticulum stress through the protein kinase C-delta-independent pathway in human colon cancer cells. Apoptosis 13:1378–1385PubMedCrossRefGoogle Scholar
  40. 40.
    Eom KS, Kim HJ, So HS, Park R, Kim TY (2010) Berberine-induced apoptosis in human glioblastoma T98G cells is mediated by endoplasmic reticulum stress accompanying reactive oxygen species and mitochondrial dysfunction. Biol Pharm Bull 33:1644–1649PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Jian-Feng Yang
    • 1
  • Jian-Guo Cao
    • 1
  • Li Tian
    • 1
  • Fei Liu
    • 1
  1. 1.Medical CollegeHunan Normal UniversityChangshaChina

Personalised recommendations