Cultivation of Escherichia coli O157:H7 isolates
Cultivation of the five isolates of E. coli O157:H7 i.e. KL-48(2), SM-25(1), SM-7(1), DS-21(4), and control isolate ATCC 43894 was initiated by culturing on lactose broth medium (LB) at 37°C, and incubated aerobically overnight. Presumptive E. coli O157 isolates were re-confirmed using E.coli O157 latex agglutination test (Ovoid, DR120M) according to the previous method [15, 16].
Isolation of Shiga-like toxin
Isolation of Shiga-like toxin was performed by culturing of isolates on Luria Bertani / LB broth (Sigma, L3022) and incubated on 37oC, 24 h, subsequently it was centrifugated at 2000 rpm for 40 min at 4oC. 5.97 g of ammonium sulfate (Sigma, A4418)15 ml was added to 15 ml of the supernatant gradually in order to obtain 65% percentage of saturation. The solution was recentrifugated at 2000 rpm for 40 min. The supernatant was removed, and the precipitate was diluted with 3 ml of sterile physiological saline, and then dialyzed at 4oC overnight. Furthermore, the toxin was sterilized by Millipore filtered with 0.22 μm filters (Corning, 431 219). The concentration of the toxin was measured by calculation of optical dencity at a wavelength of 595 nm [17, 18].
Preparation of T47D cancer cells
T47D cancer cells as a collection from integrated research and testing laboratory, Gadjah Mada University were used in this study. One ml of T47D cell maintained under standard cell culture conditions was grown as a monolayer culture in Dulbecco’s Modified Eagle Medium (DMEM) (Sigma, D6046) supplemented by 10% Newborn Calf Serum (Sigma N4887), 100 IU penicillin/ml, 100 mg/ml Streptomycin, and 50 µg fungizon (Fisher Scientific, BW17-745H). It was incubated at 37oC, in a humidified atmosphere containing 5% CO2.
Toxicity assay
The analysis of the toxicity effect in the form of cytophatic effect (CPE) among treatments and control was done according to the previous studies by measuring inhibitory concentration 50% (IC50) value of the cells. 50 µL of T47D cells were implanted into 96 well micro plate (Merck) and incubated at 5% CO2 for 24 hours to obtain confluent growth with density of 5 x 104 cells/well. Then the media were replaced with new ones, to which 50 μL of crude toxin with serial dilution was added. After 15 min of incubation at room temperature, the crude toxin was removed and monolayer cells were washed two times with Dulbecco’s Modified Eagle Medium (DMEM). 100 µL complete growth medium (DMEM with 10% Newborn calf serum, 100 IU penicillin/ml, 100 mg/ml Streptomycin, and 50 µg fungizon) was then added to the cells before they were incubated at 37oC, 5% CO2 for 24h. A positive test was shown by the amount of T47D cell lyses after incubation. At the end of incubation, the media were removed and then the cells were washed with a solution of phosphate buffer saline (PBS). 100 μL of culture media and 10 μL of MTT reagent (3-(4, 5 dimetiltiazol-2-yl) -2.5-diphenyl tetrazolium bromide) 0.5% was added to each well. Cells were incubated again for 4-6 h in 5% CO2 incubator at 37°C to form formazan. The reaction was stopped by 100 μL of MTT reagent stopper (sodium dodecyl sulfate). The cells were incubated overnight at room temperature, and then analyzed by ELISA reader at λ 550 nm [17, 18].
Cell apoptosis and necrosis assay
Apoptosis of T47D cells was determined according to the previous method with slight modification [19, 20]. An FITC-Annexin V and PI method (Invitrogen; Thermo Fisher Scientific, Inc.) was used to assess apoptosis. Briefly, 1×106 T47D cells were harvested, washed twice with cold PBS by centrifugation at 2000 rpm for 5 min, and resuspended in 100 µL binding buffer (Thermo Fisher Scientific, Inc.). A total of 100 µL Annexin V-fluorescein isothiocyanate and 2 µL PI was added to the solution. Following 10 min incubation in the dark at room temperture. 400 µL binding buffer was added to the solution and cells were analyzed using the Accuri™ C6 Flow Cytometer. The results were analyzed using CellQuest™ software 1.0 (BD Biosciences). A quadrant dot plot was used to identify whether cells were in the early or late phase of apoptosis and whether they were living or necrotic.
Cell cycle analysis with propidium iodide staining
The method was according to the previous method with slight modification [21]. The T47D cells with density 7 x 105 cells upon completion of 24 h incubation with / without IC50 of each Shiga-like toxin local isolate and control ATCC 43894. The cell cultures were washed with PBS by centrifugation at 2000 rpm for 5 min and treated with 0.1% trypsin at 37oC. The cell suspension was collected, washed once with PBS (2000 rpm, 5 min), and re-suspended for 30 min, 4oC in PBS containing 70% cold absolute ethanol for fixation and permeabilization of the cell membrane. After that, the cells were washed twice with PBS by centrifugation at 2000 rpm for 5 min, and the cells were treated with 40 µg/mL Rnase in PBS (final volume 100 ml), for 15 min at 37oC. Finally, 2 µl of PI staining solution was added to the cells, followed by 10 min incubation in the dark at room temperature. The cell cycle analysis was performed by a Fluorescence Activated Cell Sorter (FACSCalibur, Becton Dickinson, San Jose CA USA), and PI fluorescence (designated as Fl-2 Height in the histogram plots) was measured at 488 nm. Ten thousand cells were analyzed in each experiment. The percentage of cells arrest in the G0/G1, S, and G2/M phases of the cell cycle were then determined.