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Journal of Chinese Integrative Medicine ›› 2012, Vol. 10 ›› Issue (12): 1451-1459.doi: 10.3736/jcim20121218

• Original Experimental Research • Previous Articles     Next Articles

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

Mukherjee Avinaba1,Sikdar Sourav1,Bishayee Kausik1,Paul Avijit1,Ghosh Samrat1,Boujedaini Naoual2,Rahman Khuda-Bukhsh Anisur1()   

  1. 1. Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235,West Bengal,India
    2. Boiron Laboratories, Lyon 69110, France
  • Received:2012-07-27 Accepted:2012-10-15 Online:2012-12-20 Published:2018-12-15
  • Supported by:
    This work was financially supported by a grant sanctioned to Prof. A.R. Khuda-Bukhsh, Department of Zoology, University of Kalyani, India, by Boiron Laboratories, Lyon, France.

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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.
Conclusion: Ethanolic leaf extract of TO demonstrated apoptotic and antiproliferative potentials against A549 cell line.

Key words: Thuja, plant extracts, carcinoma, non-small-cell lung,cell proliferation, apoptosis, antineoplastic agents,phytogenic, in vitro

Figure 1

Effects of Thuja occidentalis ethanolic extract on viability of A549 cell line (A) and L-132 cell line (B) Cells were exposed to Thuja occidentalis ethanolic extract for 24 h at different concentrations and the cell viability was determined by MTT assay. Results are expressed as percentage of viability and data are expressed as mean±standard deviation, n= 6; *P<0.05, vs control group. MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide."

Figure 2

Effects of Thuja occidentalis ethanolic extract on cellular proliferation tested by BrdU incorporation assay Cells were exposed to three different concentration of Thuja occidentalis ethanolic extract for 6, 12 and 24 h along with incorporation of BrdU. Incorporated BrdU was labelled with anti-BrdU antibody and intensity was measured at 405 nm using PNPP as a colour developer. Data are expressed as mean±standard deviation, n=6; *P<0.05, vs normal control group. D1: 188 μg/mL; D2: 282 μg/mL; D3: 376 μg/mL; BrdU: bromodeoxyuridine; PNPP: paranitrophenylphosphate."

Figure 3

Effects of Thuja occidentalis ethanolic extract on A549 cell migration (Light microscopy, ×20) Wound-healing assays were performed to assess cell migration. Cells were untreated or treated with Thuja occidentalis ethanolic extract (< IC50 dose 188 μg/mL) for 6, 12 and 24 h. Representative photographs of treated and untreated cells are presented. Black arrows indicate migratory cells in untreated ones which found to be less in number in the treated ones."

Figure 4

Analysis of cellular morphology by a phase-contrast microscope (Light microscopy, ×20) D1: 188 μg/mL, D2: 282 μg/mL, D3: 376 μg/mL."

Figure 5

FACS analysis after annexin V-fluorescein isothiocyanate and propidium iodide dual staining assay D1: 188 μg/mL; D2: 282 μg/mL; D3: 376 μg/mL."

Figure 6

Assessment of DNA damage by Hoechst staining, comet assay, and acridine orange-ethidium bromide staining assay (Fluorescence microscopy, ×40) DNA nick generation was indicated by Hoechst 33258 (A), increasing comet tail length (B), and acridine orange-ethidium bromide fluorescence (C). Changing colour pattern of acridine orange from green (control) to slightly orange (drug-treated) indicates significant DNA fragmentation in a dose-dependent manner. White arrows indicate fragmented nuclei of the apoptotic cells. D1: 188 μg/mL; D2: 282 μg/mL; D3: 376 μg/mL."

Figure 7

Effects of TO on Bax/Bcl2 expression at protein levels Significant Bax up-regulation and Bcl2 down-regulation were observed by enzyme-linked immunosorbent assay after drug treatment for 18 h in A549 cells. Results are expressed as percentage of control and data are expressed as mean±standard deviation, n=6; *P<0.05, vs control group. D1: 188 μg/mL; D2: 282 μg/mL; D3: 376 μg/mL."

Figure 8

Caspase 3 expression analysis after drug treatment at different doses Caspase 3 gene expression was elevated after drug treatment as revealed from RT-PCR band (A) and their relative intensities (B). Caspase 3 expression at protein level was also increased as revealed from indirect ELISA analysis (C). Band intensities of RT-PCR and protein expression by ELISA assay were expressed as percentage of control and data are expressed as mean±standard deviation, n=6; *P<0.05, vs normal control group. GAPDH was used as a housekeeping gene control. D1: 188 μg/mL; D2: 282 μg/mL; D3: 376 μg/mL; RT-PCR: reverse transcription-polymerase chain reaction; ELISA: enzyme-linked immunosorbent assay; GAPDH: glyceraldehyde 3-phosphate dehydrogenase."

Figure 9

Immunoblot analysis of caspase 3 and PARP Expression of caspase 3 and PARP was increased as revealed from Western blot band (A) and their relative intensities (B). Band intensities of Western blot analysis were expressed as percentage of control and data are expressed as mean±standard deviation, n=6; *P<0.05, vs normal control group. GAPDH was used as a housekeeping gene control. D1: 188 μg/mL; D2: 282 μg/mL; D3: 376 μg/mL; PARP: poly ADP-ribose polymerase; GAPDH: glyceraldehyde 3-phosphate dehydrogenase."

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