Propolis is a natural, resinous and strongly adhesive substance that is collected from blooms and leaves of trees and plants by honeybees and is combines with pollen and enzymes secretions of bees (1). Bees use propolis as a general-purpose sealer to smooth out the internal walls of the hive and also as a protective barrier against intruders
(2). Overall, propolis is composed of 50% resin and vegetable balsam, 30% wax, 10% essential and aromatic oils, 5% pollen and 5% various other substances, including organic debris (2). Wax and organic debris are removed throughout biological processes that are usually done by ethanol extracts, and balsam thus obtained, contains the bulk of propolis bioactive constituents. Over 300 compounds among which polyphenols, terpenoides, steroids, sugars and amino acids have been identified in raw propolis. The frequency of these compounds is influenced by botanical and geographical factors, as well as by the collection of season (1,3). Propolis is considered responsible for the low presence of bacteria and moulds within the hive. The action against microorganisms is an important characteristic of propolis and humans have used it for centuries for its pharmaceutical properties (4,5). Besides its antibacterial, antifungal, antiviral characteristics, propolis presents many other biological activities including antioxidant, antiinflammatory, antitumor, hepatoprotective, local anesthetic, immunostimulatory and anti-mutagenic (2,6- 8). For this, propolis is used as a generic remedy in folk medicine, as a constituent of biocosmetics and healthy foods, and for numerous other purposes (4,5,7). Propolis is relatively non-toxic, with a non-observed effect level (NOEL) of 1400 mg/kg body weight/day in a mouse study (2).
Gastric cancer is the fourth most important cancer and the second leading cause of cancer-related deaths worldwide (9), including one million mortalities per year worldwide (10). Cancer is one of the most important healthcare problems in Iran. After road accidents and cardiovascular diseases, cancers are the third leading cause of mortalities in Iran. More than 30 000 individuals die per year because of cancer, and it is estimated that more than 70 000 individuals per year develop cancer in Iran. Out of all cancers that have been registered to date, skin cancer, breast carcinoma, gastric cancer, colorectal cancer, bladder cancer, hematopoietic system cancer, prostate cancer, esophagus cancer, lymph nodes cancer, and lungs cancer are the most prevalent. In Iran, over 50% of prevalent cancers are gastrointestinal tract-related, among which gastric cancer is the most prevalent. Most gastric cancers are developed in the elderly, and since Iranian population is relatively young, the incidence rate of and the mortality due to this fatal disease may be increase rapidly in the near future as life expectancy increases. Therefore, in light of the importance of fighting with this fatal disease, a program for controlling and treating it seems necessary (11). In the present study, anticancer activity of the ethanolic extract of propolis collected from Dinaran area (Iran) was investigated on AGS human gastric cancer cell line.
Materials and Methods
CHEMICALS AND REAGENTS
Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, and trypsin-EDTA were purchased from Gibco Co (Invitrogen, Carlsbad, CA, USA). Dymethyle sulfoxide (DMSO) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay Kit was purchased from BIO IDEA Co (Tehran-Iran).
PREPARATION OF PROPOLIS EXTRACTS
Propolis samples were collected as fresh from Dinaran area (Iran) and kept at -20°C. Ethanolic extract of propolis (EEP) was prepared according to Kalogeropoulos and Konteles method (12). Briefly, the collected propolis samples were frozen for 24 h. Then, they were grounded by moulinex vivacio grinder, and 50 g of the obtained powder was dissolved in 500 cc ethanol solution (V/V) in a dark glass container and incubated at 37°C for 14 days. The solution was shaken twice a day throughout the incubation period. After 14 time period, the obtained extract was filtered by Whatman filter paper (No. 4). To remove waxes and less soluble substances, the suspensions were subsequently frozen at -20°C for 24 hours, then filtered with Whatman (NO.4) filter paper. The freezing-filtration cycle was repeated three times. The final filtration led to represent the balsam (tincture) of propolis and is referred to as EEP (ethanolic extract of propolis). The solutions were evaporated to near dryness on a rotary evaporator (EYELA Rotary Evaporator N-100) under reduced pressure at 40°C. The remaining extract was incubated at 37°C for two weeks till the remainder of the ethanol was evaporated and the resulting powders were kept at -20°C. For experimental treatments, EEP at 100 mg/ml concentration was dissolved in DMSO as solvent and stored at -20°C.
CELL LINE AND CULTURE
AGS human gastric cancer cell-line was purchased from National Cell Bank of Iran (NCBI), Pasteur Institute of Iran (NCBI, C131). AGS cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA) in an incubator with humidified air with 5% CO2 at 37°C (13). For maintaining in the exponential growth phase, the cells were passaged at 70%-80% confluence once a week by trypsin-EDTA (Gibco, USA) (14). EEP were dissolved in DMSO as solvent and were prepared as 100 mg/ml stock solution and were kept at 100 ml volumes at -20°C. Different concentrations of EEP were prepared with DMEM containing FBS 10% just prior to AGS cells treatment.
CELL PROLIFERATION ASSAYS
Cell viability was measured by MTT assay kit (BIO IDEA Co. Tehran, Iran). The viability of AGS cells was measured in different concentrations of EEP (µg/ml) within 24, 48 and 72 hours. Cells were seeded into 96 well plates at a density of 5000 cells per well and the plates were incubated at 37°C temperature in a humidified incubator containing 5% CO2 for 24 hours. After 24 hours, the medium was removed and the cells were treated for 24, 48 and 72 hours with medium containing different concentrations (0, 10, 15, 20, 30, 40, 50, 60, 80, and 100 µg/ml) of EEP. The cells that were not treated with EEP were considered as control. The same volume of DMSO was used as the vehicle control for EEP experiments at a final concentration of 0.1%. Each EEP concentration was represented by 3 wells and replicated thrice. After 24, 48 and 72 hours, AGS cells viability was measured by MTT Assay kit according to manufacture protocol. Finally, optical density (OD) of each well was measured by an ELISA reader (AWARENESS-State Fax, USA) at 570-nm wavelength and the rate of viability (%) was calculated by the following formula:
Cell viability rate (%) = OD of treated cells/OD of control cells × 100
The statistical analysis of MTT assay data for different concentrations of EEP within 24, 48, 72 hours that calculated as viability percent was done by SPSS 19 using one-way ANOVA followed by Dennett’s test. P<0.05 was considered as statistically significant.