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.
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.