Potent cytotoxic effects of Calomeria amaranthoides on ovarian cancers Caroline van Haaften1 , Colin C Duke2 , Arij M Weerheim3 , Nico PM Smit4 , Paul MM van Haard5 , Firouz Darroudi6 and Baptist JMZ Trimbos1
Department of Gynaecology, Leiden University Medical Center, The Netherlands 2
Faculty of Pharmacy, University of Sydney, NSW 2006, Australia 3
Skin Research Laboratory, Leiden University Medical Center, Leiden, The Netherlands 4
Department of Clinical Chemistry, Leiden University Medical Center, Leiden, The Netherlands 5
Department of Clinical Chemistry, Medical Laboratories, Reinier de Graaf Group of Hospitals, Delft, The Netherlands 6
Department of Toxicogenetics, Leiden University, Medical Center Leiden, The Netherlands
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.
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.
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.
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.