title: Ethanolic extract of Thuja occidentalis blocks proliferation of A549 cells and induces apoptosis in vitro.
abstract: OBJECTIVE
  To study the possible anticancer and antiproliferative activities of ethanolic leaf extract of Thuja occidentalis (TO) on A549 non-small lung carcinoma cells in vitro.
  
  
  METHODS
  Cell viability was ascertained through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay after deployment of TO in different doses. The half maximal inhibitory concentration (IC50) dose (282 μg/mL) was determined, and two other doses for dose-dependence study, one below the IC50 dose (IC35=188 μg/mL) and one above the IC50 dose (IC65=376 μg/mL) were selected. Bromodeoxyuridine (BrdU) incorporation assay and migration studies were performed to elucidate antiproliferative activity of the drug, if any. Fluorescence-activated cell sorting analysis after annexin V-fluorescein isothiocyanate and propidium iodide (annexin V-FITC-PI) dual staining method was done to ascertain early stage of apoptosis, if any. DNA fragmentation assay was done through Hoechst 33258 and acridine orange-ethidium bromide staining. DNA damage was quantified through comet assay. Bax-Bcl2 regulation and expression studies were performed through indirect enzyme-linked immunosorbent assay (ELISA). Caspase 3 activity was measured at gene level through reverse transcription-polymerase chain reaction (RT-PCR) analysis. Its activation at protein level was analyzed through indirect ELISA and Western blot analysis.
  
  
  RESULTS
  TO demonstrated a dose-dependent decrease in viability of A549 cells after 24 h of exposure. Cell proliferation was reduced in a time-dependent manner of drug exposure as revealed from BrdU incorporation and migration studies. Annexin-V-FITC positivity of cells up to 11.72% as compared to the untreated control revealed early state of TO-induced apoptosis. Occurrence of comet tail and increased fluorescence of Hoechst after 24 h of drug exposure revealed significant DNA nick generation and chromatin condensation. Bax up-regulation and Bcl-2 down-regulation suitably altered ratio of Bax/Bcl-2 in favor of apoptosis. From RT-PCR, indirect ELISA and Western blot studies, caspase 3 activity was also found to be significantly increased along with cleaved poly ADP-ribose polymerase expression.
  
  
  CONCLUSIONS
  Ethanolic leaf extract of TO demonstrated apoptotic and antiproliferative potentials against A549 cell line.
authors.affiliation: Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, West Bengal, India.
authors.forename: Avinaba
authors.initials: A
authors.lastname: Mukherjee
authors.forename: Sourav
authors.initials: S
authors.lastname: Sikdar
authors.forename: Kausik
authors.initials: K
authors.lastname: Bishayee
authors.forename: Avijit
authors.initials: A
authors.lastname: Paul
authors.forename: Samrat
authors.initials: S
authors.lastname: Ghosh
authors.forename: Naoual
authors.initials: N
authors.lastname: Boujedaini
authors.forename: Anisur Rahman
authors.initials: AR
authors.lastname: Khuda-Bukhsh
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D006801:Humans; D010936:Plant Extracts / Q000494:pharmacology*; D018515:Plant Leaves / Q000737:chemistry; D029783:Thuja / Q000737:chemistry*
chemical_list: D010936:Plant Extracts