Cell-free supernatant preparation
Five species of bifidobacteria including B. adolescentis (ATCC 15703, PTCC1536), B. animalis subsp. lactis (PTCC1736), B. animalis subsp. animalis (ATCC 25527, PTCC 1631), B. bifidum (ATCC 29521, PTCC 1644), and B. angulatum (ATCC 27535, PTCC 1366) were obtained from the Persian Type Culture Collection (PTCC) from the Iranian Research Organization for Science and Technology (IROST). Bifidobacterial strains were cultured in de Man–Rogosa agar (MRS) (Merck, Darmstadt, Germany) at 37 °C for 72 hours in an anaerobic incubator. Then, the cultures were centrifuged at 4500 rpm for 15 min at 4 °C. Fifty ml of each supernatant mixed with 75 ml volume of methanol and gently agitated for 24 h. The methanolic extracts were dried by lyophilization and obtained dried materials were dissolved in different amounts (5–30 mg/ml) in each used cell culture including Dulbecco Modified Eagle medium (DMEM) (Gibco, Grand Island, NY, USA), and Roswell Park Memorial Institute medium (RPMI) 1640 (Sigma, St Louis, MO, USA). Finally, the pH of supernatants was adjusted to 7.2 points and before using, filtered through a 0.22 mm Millipore filter (Milli-Q, Millipore, Germany).
Cell Culture
Two human colon cancer cell lines (Caco-2, ATCC, HTB-37 and HT-29, ATCC, HTB-38) and one human epithelial normal cell line with the same embryonic origin (KDR/293) were purchased from Pasteur Institute (national cell bank of Iran). The purchased cells were cultured in 25 cm2 plastic cell culture flasks and were incubated under standard conditions at 37˚C in a humidified atmosphere with 5% CO2 with medium renewal every 1–3 days. The cells were maintained in an RPMI-1640 (HT-29 and Caco-2) or high glucose concentration (4.5 g/l) DMEM (KDR/293) cell culture medium, respectively. Both media were supplemented with 10% (v/v) fetal bovine serum (FBS) (HyClone, Logan, UT, USA), 8 mM L-glutamine and 1% of mixture penicillin (100 IU/ml) and streptomycin (100 g/ml) (Sigma, St Louis, MO, USA).
Mtt Assay
Cell viability in treated and untreated cell lines was determined by the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma, St Louis, MO, USA) based on the capacity of viable cells to reduce a tetrazolium colorless salt to purple formazan in mitochondria. At first, the half-maximal inhibitory concentration (IC50) for HT-29 and Caco-2 cells was determined using prescreening MTT tests (in the range of 10 to 100 µg/mL) at 24 and 48 hours. Briefly, the cells were washed twice with phosphate-buffered saline (PBS) (Sigma, St Louis, MO, USA) and trypsinized by adding 1 ml of trypsin/EDTA (Sigma, St Louis, MO, USA) solution. The cells were plated into 96well plates at 1.2 × 104 cells per well and added 200 µL of the growth medium, incubated for 24-h. After cell attachment, the medium was carefully removed from each well and the cells were treated with an effective dose (IC-50) of cell-free bacterial supernatant and growth medium. After 24 h or 48 h (determined time point) incubation, 50 µl of MTT reagent and 150 µL of fresh growth medium were added to each well and plates returned to the incubator for 4 hours. Then, the medium of each well was carefully removed and 200 µL of dimethyl sulfoxide (Merck, Germany) and 25 µL of Sorenson buffer (0.1 mol/L glycine, 0.1 mol/L NaCl, pH 10.5) were added to each well and kept for 15 minutes in the dark condition at room temperature. The absorbance was determined using enzyme-linked immune-sorbent assay plate reader (ELx 800; Biotek, Winooski, VT, USA) at 570 nm. The growth inhibitory effects of supernatant were calculated according to the following formula: the growth inhibition ratio = [(the absorbance of blank control group – the absorbance of experimental group)/the absorbance of blank control group] × 100% (22, 23). In the present study, 5-fluorouracil (5-FU) as approved anticancer drug (7 µL/well of 96-well plate) was used as the positive control.
Flow Cytometry
Three mL of growth medium including 1.2 × 105 cells was cultured in 6-well culture plates and incubated at growth condition. After 24 h, the cells were treated with 3 ml of the sterile growth medium containing determined dried materials of supernatant or 5-FU, as the positive control group, and incubated in the growth condition based on the determined time point. The treated/untreated control cells were detached by trypsin-EDTA, and supernatants were discarded by centrifugation at 900 rpm for 10 minutes. Finally, for detection of apoptosis, the cells were stained with Annexin V-FITC/propidium iodide (PI) apoptosis kit (eBioscience, San Diego, CA, USA) according to the manufacturer's instructions, and data analysis was conducted using CELL Quest Pro software (BD Biosciences, San Jose, CA, USA). Each experiment was repeated 2 times with triplicate samples. All of the analyses were performed using 150000 cells at a rate of 900 cell/sec.
Quantitative Real-time Pcr Analysis
All untreated/treated cells were washed three times with PBS (pH 7.2) and total RNA was extracted from cells by direct lysis using 1 ml ice cold RNX-plus solution (Sina Clone, Iran), according to manufacturer's instruments. The obtained total RNA was solved in 50 µL DEPC-treated water (Merck, Germany), and the quantity and quality of total RNA were evaluated by UV spectrophotometry and agarose gel electrophoresis, respectively. Complementary DNA (cDNA) was synthesized using one microgram of isolated RNA by Prime Script RT Reagent kit (Takara Bio Inc, Tokyo, Japan) according to the manufacturer's instructions. The specific primers for every gene including Bcl-2, BAD, Fas-R, caspase-3, caspase-8, caspase-9, and GAPDH as housekeeping gene were designed (10). All of the amplification reactions were carried out in triplicate for each sample, and every experiment mixture (20 µL), containing 10 µL SYBR Green PCR master mix (Takara Bio Inc, Tokyo, Japan), 1 µL cDNA (1 µg/µL), 1 µL primer (forward and reverse), and 0.8 µL 6-carboxy-X-rhodamine (ROX as reference dye), was subjected to ABI-step I plus (Applied Biosystems, Foster City, CA, USA) instrument. One cycle at 95 °C for 5 minutes followed by 40 cycles at 95 °C for 20 seconds, 60 °C for 35 seconds, and 72 °C for 10 seconds were selected as thermal cycling condition. Pfaffle method was used for interpretation of the results and the threshold cycle values were normalized to the expression rate of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (24).
Statistical analysis
The statistical package for the social sciences (SPSS Inc. Chicago, IL, USA version 16.0) was used for the statistical analysis. One-way ANOVA and Tukey’s post hoc test were performed for analyzing differences between all treatments and multiple mean comparisons, respectively. Statistical significance was considered to be P ≤ 0.05.