title: Antiproliferative and cytostatic effects of the natural product eupatorin on MDA-MB-468 human breast cancer cells due to CYP1-mediated metabolism.
abstract: BACKGROUND
  The natural product eupatorin has been reported to have antiproliferative activity in tumour cell lines, but the exact mechanism is unclear. The cytochromes P450 CYP1B1, CYP1A1, and CYP1A2 have been shown to participate in the activation of various xenobiotics, compounds derived from the diet as well as chemotherapeutic drugs. CYP1B1 and CYP1A1 have also been proposed as targets for cancer chemotherapy for their differential and selective overexpression in tumour cells. In this study, we aimed to identify a possible mechanism of action for the antiproliferative effect of eupatorin, which can be attributed to CYP1 family-mediated metabolism.
  
  
  METHODS
  The study focuses on the antiproliferative action of eupatorin on the human breast carcinoma cell line MDA-MB-468 and on a cell line derived from normal mammary tissue, MCF-10A. The cytotoxicity of the flavone, its effect on the cell cycle of the abovementioned cell lines, and its metabolism by CYP1 family enzymes were examined.
  
  
  RESULTS
  Eupatorin showed a dose-dependent inhibitory effect of cell growth on MDA-MB-468 cells with a submicromolar median inhibition concentration (IC50) whereas the IC50 of this compound in MCF-10A cells was considerably higher. The antiproliferative effect, as measured by EROD (ethoxyresorufin-O-deethylase) assay and Western immunoblotting, was attributed mainly to CYP1A1 expression in MDA-MB-468 cells but not in MCF-10A cells. Moreover, CYP1 family enzymes were shown to metabolise eupatorin in vitro to the flavone cirsiliol and two other unidentified metabolites. Metabolism of eupatorin was also detected in MDA-MB-468 cell cultures, whereas metabolism by MCF-10A cells was negligible. Eupatorin was further shown to arrest the cell cycle of the CYP1-expressing cell line MDA-MB-468 in G2/M phase, whereas no effect was observed in MCF-10A cells, which do not express CYP1 enzymes. The effect of eupatorin on the MDA-MB-468 cell cycle could be reversed by co-application of the CYP1 inhibitor acacetin.
  
  
  CONCLUSIONS
  The flavone eupatorin is selectively activated in breast cancer cells, but not in normal breast cells, due to CYP1 family metabolism. This provides a basis for selectivity which is desired against breast tumour cells. In this sense, eupatorin is shown by this study to be a very promising chemopreventative candidate that should be examined further in an in vivo study.
authors.affiliation: Leicester School of Pharmacy, De Montfort University, The Gateway, Leicester, UK.
authors.forename: Vasilis
authors.initials: V
authors.lastname: Androutsopoulos
authors.forename: Randolph R J
authors.initials: RR
authors.lastname: Arroo
authors.forename: John F
authors.initials: JF
authors.lastname: Hall
authors.forename: Somchaiya
authors.initials: S
authors.lastname: Surichan
authors.forename: Gerry A
authors.initials: GA
authors.lastname: Potter
mesh_terms: D016588:Anticarcinogenic Agents / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D001940:Breast / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism*; D019388:Cytochrome P-450 CYP1A2 / Q000378:metabolism*; D065633:Cytochrome P-450 CYP1B1; D005419:Flavonoids / Q000494:pharmacology*; D006801:Humans; D020128:Inhibitory Concentration 50; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology
chemical_list: D016588:Anticarcinogenic Agents; D000970:Antineoplastic Agents; D005419:Flavonoids; D013778:Tetrazolium Salts; D013844:Thiazoles; C103110:eupatorin; D001189:Aryl Hydrocarbon Hydroxylases; C585496:CYP1B1 protein, human; D019363:Cytochrome P-450 CYP1A1; D019388:Cytochrome P-450 CYP1A2; D065633:Cytochrome P-450 CYP1B1; C022616:thiazolyl blue