title: Induction of non-apoptotic cell death in human oral squamous cell carcinoma cell lines by Rhinacanthus nasutus extract.
abstract: BACKGROUND
  We recently reported that the MeOH extract of aerial parts and root of Rhinacanthus nasutus showed diverse biological activity, with most activity being concentrated into the EtOAc layer separated by sequential organic solvent extractions. In the present study, the EtOAc extracts were further separated by silica-gel column chromatography into five fractions (Frs. 1-5), and their cytotoxicity and apoptosis-inducing activity investigated.
  
  
  METHODS
  Cytotoxic activity was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) method. The 50% cytotoxic concentration (CC(50)) was determined from the dose-response curve. Tumor specificity (TS) was determined by the ratio of the mean CC(50) for normal cells to the one for tumor cell lines. DNA fragmentation was assayed by agarose gel electrophoresis. Caspase-3/-7 activation was monitored by cleavage of substrates either spectrophotometrically or by western blot analysis.
  
  
  RESULTS
  Among five fractions of the EtOAc extract, Fr. 1, eluted with CHCl(3)-MeOH (50:1), showed the highest tumor specificity (TS=3.3) as compared with other fractions eluted at higher concentrations of MeOH in CHCl(3) (TS=1.0-2.8). Fr. 1 did not induce internucleosomal DNA fragmentation or induced only marginal level of caspase-3 activity in either human promyelocytic leukemia HL-60 cells and human oral squamous cell carcinoma (OSCC) cell lines HSC-2.
  
  
  CONCLUSIONS
  The present study suggests that hydrophobic substances of EtOAc extract show tumor specific cytotoxicity by inducing little or no apoptosis.
authors.affiliation: Faculty of Pharmaceutical Sciences, Josai University, Sakado, Saitama Japan.
authors.forename: Haruka
authors.initials: H
authors.lastname: Horii
authors.forename: Ryuichiro
authors.initials: R
authors.lastname: Suzuki
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sakagami
authors.forename: Naoki
authors.initials: N
authors.lastname: Umemura
authors.forename: Jun-ya
authors.initials: JY
authors.lastname: Ueda
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Shirataki
mesh_terms: D000085:Acetates; D000972:Antineoplastic Agents, Phytogenic / Q000302:isolation & purification / Q000494:pharmacology*; D002294:Carcinoma, Squamous Cell / Q000473:pathology*; D053148:Caspase 3 / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor / Q000187:drug effects; D002850:Chromatography, Gel; D053938:DNA Fragmentation / Q000187:drug effects; D004273:DNA, Neoplasm / Q000187:drug effects; D004354:Drug Screening Assays, Antitumor; D004789:Enzyme Activation / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects; D005347:Fibroblasts / Q000187:drug effects; D018922:HL-60 Cells / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D019684:Magnoliopsida / Q000737:chemistry*; D009062:Mouth Neoplasms / Q000473:pathology*; D010936:Plant Extracts / Q000494:pharmacology*; D018517:Plant Roots / Q000737:chemistry; D018547:Plant Stems / Q000737:chemistry; D012997:Solvents
chemical_list: D000085:Acetates; D000972:Antineoplastic Agents, Phytogenic; D004273:DNA, Neoplasm; D010936:Plant Extracts; D012997:Solvents; C007650:ethyl acetate; D053148:Caspase 3