title: Pro-apoptotic effects of tectorigenin on human hepatocellular carcinoma HepG2 cells.
abstract: OBJECTIVE
  To investigate the effects of tectorigenin on human hepatocellular carcinoma (HCC) HepG2 cells.
  
  
  METHODS
  Tectorigenin, one of the main components of rhizome of Iris tectorum, was prepared by simple methods, such as extraction, filtration, concentration, precipitation and recrystallization. HepG2 cells were incubated with tectorigenin at different concentrations, and their viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was detected by morphological observation of nuclear change, agarose gel electrophoresis of DNA ladder, and flow cytometry with Hoechst 33342, Annexin V-EGFP and propidium iodide staining. Generation of reactive oxygen species was quantified using DCFH-DA. Intracellular Ca(2+) was monitored by Fura 2-AM. Mitochondrial membrane potential was monitored using Rhodamine 123. Release of cytochrome c from mitochondria to cytosol was detected by Western blotting. Activities of caspase-3, -8 and -9 were investigated by Caspase Activity Assay Kit.
  
  
  RESULTS
  The viability of HepG2 cells treated by tectorigenin decreased in a concentration- and time-dependent manner. The concentration that reduced the number of viable HepG2 cells by 50% (IC(50)) after 12, 24 and 48 h of incubation was 35.72 mg/L, 21.19 mg/L and 11.06 mg/L, respectively. However, treatment with tectorigenin at 20 mg/L resulted in a very slight cytotoxicity to L02 cells after incubation for 12, 24 or 48 h. Tectorigenin at a concentration of 20 mg/L greatly inhibited the viability of HepG2 cells and induced the condensation of chromatin and fragmentation of nuclei. Tectorigenin induced apoptosis of HepG2 cells in a time- and dose-dependent manner. Compared with the viability rate, induction of apoptosis was the main mechanism of the anti-proliferation effect of tectorigenin in HepG2 cells. Furthermore, tectorigenin-induced apoptosis of HepG2 cells was associated with the generation of reactive oxygen species, increased intracellular [Ca(2+)](i), loss of mitochondrial membrane potential, translocation of cytochrome c, and activation of caspase-9 and -3.
  
  
  CONCLUSIONS
  Tectorigenin induces apoptosis of HepG2 cells mainly via mitochondrial-mediated pathway, and produces a slight cytotoxicity to L02 cells.
authors.affiliation: The First Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing 210046, Jiangsu Province, China.
authors.forename: Chun-Ping
authors.initials: CP
authors.lastname: Jiang
authors.forename: Hui
authors.initials: H
authors.lastname: Ding
authors.forename: Da-Hua
authors.initials: DH
authors.lastname: Shi
authors.forename: Yu-Rong
authors.initials: YR
authors.lastname: Wang
authors.forename: Er-Guang
authors.initials: EG
authors.lastname: Li
authors.forename: Jun-Hua
authors.initials: JH
authors.lastname: Wu
mesh_terms: D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D045304:Cytochromes c / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D007498:Iris / Q000737:chemistry; D007529:Isoflavones / Q000494:pharmacology*; D020782:Matrix Metalloproteinases / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D007529:Isoflavones; D017382:Reactive Oxygen Species; C120039:tectorigenin; D045304:Cytochromes c; D020782:Matrix Metalloproteinases; D002118:Calcium