Shahab Bohlooli1, Shahriar Bohlooli2, Roghaye Aslanian3, Fatemeh Nouri2, Ali Teimourzadeh4. 1. Department of Pharmacology and Toxicology, School of Pharmacy, Ardabil University of Medical Sciences, Ardabil, Iran; Drug and Advanced Sciences Research Center, School of Pharmacy, Ardabil University of Medical Sciences, Ardabil, Iran. Electronic address: shahab.bohlooli@arums.ac.ir. 2. Department of Pharmacology and Toxicology, School of Pharmacy, Ardabil University of Medical Sciences, Ardabil, Iran. 3. Department of Pharmacology and Toxicology, School of Pharmacy, Ardabil University of Medical Sciences, Ardabil, Iran; Drug and Advanced Sciences Research Center, School of Pharmacy, Ardabil University of Medical Sciences, Ardabil, Iran; School of Agriculture, University of Mohaghegh Ardabili, Ardabil, Iran. 4. School of Agriculture, University of Mohaghegh Ardabili, Ardabil, Iran.
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE: A weed plant belonging to the Caryophyllaceae family, Agrostemma githago is used in folk medicine to treat cancers and warts. AIM OF THE STUDY: The aim of this study was to evaluate the cytotoxic effect of the aqueous extract of A. githago seed on gastric cancer cell line (AGS) and to investigate the mechanism of apoptosis induction in these cells. MATERIALS AND METHODS: Seeds of A. githago were collected from the suburban area of Ardabil Province, northwest Iran. After preparing the aqueous extract, dry matter was harvested with the lyophilizing technique. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess the cytotoxicity. Apoptotic cells were detected by staining with ethidium bromide/acridine orange (EB/AO). Diamidino-2-phenylindole (DAPI) staining was used for cell-cycle analysis with a flow cytometer. The annexin V binding level, caspase-3 activity, and B-cell lymphoma-2 (Bcl-2) level were also measured to confirm apoptotic cell death. RESULTS: After the aqueous extract of A. githago seed was incubated for 24, 48, and 72 h, inhibited cell growth was observed with IC50 values of 13.51 ± 0.7, 4.37 ± 1.01, and 2.42 ± 0.8 µg/ml, respectively. The EB/AO staining method demonstrated that the extract exerts its cytotoxic effect mainly via apoptosis, in accordance with the annexin V, blc-2, and caspase-3 results. The extract showed a concentration-dependent increase in annexin V binding to externally exposed phosphatidylserine as well as caspase-3 activity. The bcl-2 protein level showed a proportionate decrease with the increase in extract concentration. The cell-cycle analysis revealed that the extract can arrest cells at the G1 checkpoint. CONCLUSION: The findings of this study revealed the cytotoxic effect of the aqueous extract of A. githago seed on gastric cancer cells (AGS) mainly via apoptosis and the cell-cycle arrest at the G1 checkpoint. Therefore, the extract can be potentially used in gastric cancer therapy in vitro.
ETHNOPHARMACOLOGICAL RELEVANCE: A weed plant belonging to the Caryophyllaceae family, Agrostemma githago is used in folk medicine to treat cancers and warts. AIM OF THE STUDY: The aim of this study was to evaluate the cytotoxic effect of the aqueous extract of A. githago seed on gastric cancer cell line (AGS) and to investigate the mechanism of apoptosis induction in these cells. MATERIALS AND METHODS: Seeds of A. githago were collected from the suburban area of Ardabil Province, northwest Iran. After preparing the aqueous extract, dry matter was harvested with the lyophilizing technique. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess the cytotoxicity. Apoptotic cells were detected by staining with ethidium bromide/acridine orange (EB/AO). Diamidino-2-phenylindole (DAPI) staining was used for cell-cycle analysis with a flow cytometer. The annexin V binding level, caspase-3 activity, and B-cell lymphoma-2 (Bcl-2) level were also measured to confirm apoptotic cell death. RESULTS: After the aqueous extract of A. githago seed was incubated for 24, 48, and 72 h, inhibited cell growth was observed with IC50 values of 13.51 ± 0.7, 4.37 ± 1.01, and 2.42 ± 0.8 µg/ml, respectively. The EB/AO staining method demonstrated that the extract exerts its cytotoxic effect mainly via apoptosis, in accordance with the annexin V, blc-2, and caspase-3 results. The extract showed a concentration-dependent increase in annexin V binding to externally exposed phosphatidylserine as well as caspase-3 activity. The bcl-2 protein level showed a proportionate decrease with the increase in extract concentration. The cell-cycle analysis revealed that the extract can arrest cells at the G1 checkpoint. CONCLUSION: The findings of this study revealed the cytotoxic effect of the aqueous extract of A. githago seed on gastric cancer cells (AGS) mainly via apoptosis and the cell-cycle arrest at the G1 checkpoint. Therefore, the extract can be potentially used in gastric cancer therapy in vitro.