title: Potent cytotoxic effects of Calomeria amaranthoides on ovarian cancers.
abstract: BACKGROUND
  Ovarian cancer remains the leading cause of death from gynaecological malignancy. More than 60% of the patients are presenting the disease in stage III or IV. In spite of combination of chemotherapy and surgery the prognosis stays poor for therapy regimen.
  
  
  METHODS
  The leaves of a plant endemic to Australia, Calomeria amaranthoides, were extracted and then fractionated by column chromatography. In vitro cytotoxicity tests were performed with fractions of the plant extract and later with an isolated compound on ovarian cancer cell lines, as well as normal fibroblasts at concentrations of 1-100 μg/mL (crude extract) and 1-10 μg/mL (compound). Cytotoxicity was measured after 24, 48 and 72 hours by using a non-fluorescent substrate, Alamar blue.In vivo cytotoxicity was tested on ascites, developed in the abdomen of nude mice after inoculation with human OVCAR3 cells intraperitoneally. The rate of change in abdomen size for the mice was determined by linear regression and statistically evaluated for significance by the unpaired t test.
  
  
  RESULTS
  Two compounds were isolated by chromatographic fractionation and identified by 1H-NMR, 13C-NMR and mass spectrometry analyses, EPD, an α-methylene sesquiterpene lactone of the eremophilanolide subtype, and EPA, an α-methylene carboxylic acid.Cytotoxicity of EPD for normal fibroblasts at all time points IC50 was greater than 10 μg/mL, whereas, for OVCAR3 cells at 48 hours IC50 was 5.3 μg/mL (95% confidence interval 4.3 to 6.5 μg/mL).Both, the crude plant extract as well as EPD killed the cancer cells at a final concentration of 10 μg/mL and 5 μg/mL respectively, while in normal cells only 20% cell killing effect was observed. EPA had no cytotoxic effects.Changes in abdomen size for control versus Cisplatin treated mice were significantly different, P = 0.023, as were control versus EPD treated mice, P = 0.025, whereas, EPD versus Cisplatin treated mice were not significantly different, P = 0.13.
  
  
  CONCLUSIONS
  For the first time both crude plant extract from Calomeria amaranthoides and EPD have been shown to have potent anti-cancer effects against ovarian cancer.
authors.affiliation: Department of Gynaecology, Leiden University Medical Center, The Netherlands. carocell@planet.nl
authors.forename: Caroline
authors.initials: C
authors.lastname: van Haaften
authors.forename: Colin C
authors.initials: CC
authors.lastname: Duke
authors.forename: Arij M
authors.initials: AM
authors.lastname: Weerheim
authors.forename: Nico Pm
authors.initials: NP
authors.lastname: Smit
authors.forename: Paul Mm
authors.initials: PM
authors.lastname: van Haard
authors.forename: Firouz
authors.initials: F
authors.lastname: Darroudi
authors.forename: Baptist Jmz
authors.initials: BJ
authors.lastname: Trimbos
mesh_terms: D000179:Acrylates / Q000737:chemistry / Q000627:therapeutic use*; D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000627:therapeutic use*; D019659:Asteraceae / Q000737:chemistry*; D001315:Australia; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002945:Cisplatin / Q000627:therapeutic use; D005260:Female; D006801:Humans; D007783:Lactones / Q000737:chemistry / Q000627:therapeutic use*; D051379:Mice; D008819:Mice, Nude; D010051:Ovarian Neoplasms / Q000188:drug therapy*; D010078:Oxazines; D010936:Plant Extracts / Q000737:chemistry / Q000627:therapeutic use*; D012717:Sesquiterpenes / Q000737:chemistry / Q000627:therapeutic use*; D014966:Xanthenes
chemical_list: D000179:Acrylates; D000970:Antineoplastic Agents; D007783:Lactones; D010078:Oxazines; D010936:Plant Extracts; D012717:Sesquiterpenes; D014966:Xanthenes; C558918:eremophila-1(10),11(13)-dien-12-oic acid; C558919:eremophila-1(10)-11(13)-dien-12,8-olide; C005843:resazurin; D002945:Cisplatin