title: Cytotoxicity of Eupatorium cannabinum L. ethanolic extract against colon cancer cells and interactions with Bisphenol A and Doxorubicin.
abstract: BACKGROUND
  Eupatorium cannabinum L. has long been utilized in traditional medicine, however no information is available regarding cellular effects of full extracts. Here we assessed the effects of E. cannabinum ethanolic extract (EcEE) on the colon cancer line HT29. Potential interactions with bisphenol A (BPA) a synthetic phenolic compound to which humans are generally exposed and a commonly used chemotherapeutic agent, doxorubicin (DOX) were also evaluated.
  
  
  METHODS
  HT29 cells were exposed to different concentrations (0.5 to 50 μg/ml) of EcEE alone or in combination with BPA or DOX. Cell viability was analyzed through resazurin assay. Gene transcription levels for NCL, FOS, p21, AURKA and bcl-xl were determined through qRT-PCR. Cytological analysis included evaluation of nuclear and mitotic anomalies after DAPI staining, immunodetection of histone H3 lysine 9 acetylation (H3K9ac) and assessment of DNA damage by TUNEL assay.
  
  
  RESULTS
  Severe loss of HT29 cell viability was detected for 50 μg/ml EcEE immediately after 24 h exposure whereas the lower concentrations assayed (0.5, 5 and 25 μg/ml) resulted in significant viability decreases after 96 h. Exposure to 25 μg/ml EcEE for 48 h resulted in irreversible cell damage leading to a drastic decrease in cell viability after 72 h recovery in EcEE-free medium. 48 h 25 μg/ml EcEE treatment also induced alteration of colony morphology, H3K9 hyperacetylation, transcriptional up regulation of p21 and down regulation of NCL, FOS and AURKA, indicating reduced proliferation capacity. This treatment also resulted in drastic mitotic and nuclear disruption accompanied by up-regulation of bcl-xl, limited TUNEL labeling and nuclear size increase, suggestive of a non-apoptocic cell death pathway. EcEE/BPA co-exposure increased mitotic anomalies particularly for the lowest EcEE concentration, although without major effects on viability. Conversely, EcEE/DOX co-exposure decreased cell viability in relation to DOX for all EcEE concentrations, without affecting the DOX-induced cell cycle arrest.
  
  
  CONCLUSIONS
  EcEE has cytotoxic activity on HT29 cancer cells leading to mitotic disruption and non-apoptotic cell death without severe induction of DNA damage. Interaction experiments showed that EcEE can increase BPA aneugenic effects and EcEE synergistic effects with DOX supporting a potential use as adjuvant in chemotherapeutic approaches.
authors: Edna
authors: E
authors: Ribeiro-Varandas
authors: Filipe
authors: F
authors: Ressurreição
authors: Wanda
authors: W
authors: Viegas
authors: Centro de Botânica Aplicada à Agricultura, Instituto Superior de Agronomia, Universidade de Lisboa, Tapada da Ajuda, 1349-017 Lisboa, Portugal. mxdelgado@isa.ulisboa.pt.
authors: Margarida
authors: M
authors: Delgado
mesh_terms: D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D001559:Benzhydryl Compounds / Q000008:administration & dosage / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000473:pathology; D004317:Doxorubicin / Q000008:administration & dosage / Q000494:pharmacology*; D004357:Drug Synergism; D036602:Eupatorium / Q000737:chemistry*; D019073:HT29 Cells; D006801:Humans; D008938:Mitosis / Q000187:drug effects; D010636:Phenols / Q000008:administration & dosage / Q000494:pharmacology*; D010936:Plant Extracts / Q000494:pharmacology*; D000098826:Bisphenol A Compounds
chemical_list: D001559:Benzhydryl Compounds; D004317:Doxorubicin; D010636:Phenols; D010936:Plant Extracts; C006780:bisphenol A; D000098826:Bisphenol A Compounds