title: 5, 7-Dimethoxyflavone sensitizes TRAIL-induced apoptosis through DR5 upregulation in hepatocellular carcinoma cells.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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).
  
  
  CONCLUSIONS
  The present study demonstrates that DMF selectively enhances TRAIL-induced apoptosis by ROS-stimulated ER-stress triggering CHOP-mediated DR5 upregulation in HCC.
authors.affiliation: Medical College, Hunan Normal University, Changsha 410013, China.
authors.forename: Jian-Feng
authors.initials: JF
authors.lastname: Yang
authors.forename: Jian-Guo
authors.initials: JG
authors.lastname: Cao
authors.forename: Li
authors.initials: L
authors.lastname: Tian
authors.forename: Fei
authors.initials: F
authors.lastname: Liu
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000473:pathology; D002460:Cell Line; D045744:Cell Line, Tumor; D004721:Endoplasmic Reticulum / Q000378:metabolism; D000091342:Endoplasmic Reticulum Chaperone BiP; D005419:Flavonoids / Q000494:pharmacology*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000473:pathology; D034741:RNA, Small Interfering / Q000008:administration & dosage; D017382:Reactive Oxygen Species / Q000378:metabolism; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000378:metabolism*; D051742:Transcription Factor CHOP / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D000091342:Endoplasmic Reticulum Chaperone BiP; D005419:Flavonoids; C000717247:HSPA5 protein, human; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D053221:TNF-Related Apoptosis-Inducing Ligand; D051742:Transcription Factor CHOP; C060298:5,7-dimethoxyflavone