Ethanolic extract of Thuja occidentalis blocks proliferation of A549 cells and induces apoptosis in vitro

Avinaba Mukherjee, Sourav Sikdar, Kausik Bishayee, Avijit Paul, Samrat Ghosh, Naoual Boujedaini, Anisur Rahman Khuda-Bukhsh

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

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 = 3 7 6 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. CONCLUSION: Ethanolic leaf extract of TO demonstrated apoptotic and antiproliferative potentials against A549 cell line.

Original languageEnglish
Pages (from-to)1451-1459
Number of pages9
JournalJournal of integrative medicine
Volume10
Issue number12
DOIs
StatePublished - 1 Dec 2012
Externally publishedYes

Keywords

  • Antineoplastic agents
  • Apoptosis
  • Carcinoma
  • Cell proliferation
  • In vitro
  • Non-small-cell lung
  • Phytogenic
  • Plant extracts
  • Thuja

ASJC Scopus subject areas

  • Complementary and alternative medicine

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