br This study used breast cancer
This study used breast cancer cell lines the MCF-7 as the experi-mental cancer VH-298 (ATCC No: HTB-22™), and VERO cell line (ATCC® No: CCL-81™) as a control cell line. These cells were obtained from American type Collection Culture. Frozen cells were thawed and in-oculated into 5 mL of Dulbecco's Modification of Eagle's Medium (DMEM) supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum (FBS). The cells were cultured in T-25 flasks and in-cubated in 95% humidified incubator with 5% CO2 at 37 °C. The cell cultures were used when reached approximately 70% confluence for experimental treatments.
2.5. Cell viability assay (cytotoxicity assay)
The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was used to quantify the percentage of cell viability. The cultured cells were detached with accutase, suspended in media and counted using a hemocytometer. Approximately 50,000 cells were plated in 100 μL of medium per well in 96-well plate then incubated for 24 h in 5% CO2 incubator. The cells were then treated (triplicate wells per condition) by adding 20 μL of serial dilutions of the sample extract in DMEM free-fetal bovine serum (the control samples received only DMSO). Then incubate the cells for another 24 h before adding 20 μL (5 mg/mL) solution of MTT reagent into each well. Then the incubation was continued for another 3 h before the media was pipetted out carefully. 100 μL of solubilization solution (DMSO) was added to each well and mixed by gentle shaking for 15 min (Liu et al., 2016). Finally, the amount of formazan was determined by measuring the absorbance at 570 nm using a microplate reader. The obtained data were used to calculate the percentage of viable/dead cells, and fifty percent of in-hibitory concentration (IC50) of each extract was determined, using equation (1):
Abs of control
where Abs of the sample is the absorbance of treated cells, and Abs of control is the absorbance of untreated cells.
D. Daddiouaissa, et al.
2.6. Estimation of the inhibition concentration at 50% (IC50) Coulter, USA). The cells in S and G2M phases are defined as pro-
liferative cells and as apoptotic cells in the sub-G1 phase (Agu et al.,
The IC50 values were graphically obtained by plotting the percen- 2018).
tage of cell viability against the corresponding of the IL-GFE sample
used. Data were reported as the mean ± standard error (SE) and sta- 2.10. Annexin-V-FITC assay
tically analyzed. The significance was accepted when P ≤0.05 level.
The Annexin -V-FITC methods were conducted using the early, and
2.7. Preparation of ionic liquid-graviola fruit extract (IL-GFE) and taxol late apoptotic cells after treatment with IL-GFE was determined using
buﬀered saline). Taxol was used as a positive control (+ve), and 50 μg removed, washed with PBS and centrifuged at 1500 rpm for 5 min.
of Taxol was dissolved in DMSO.
Then, cells were resuspended in the binding buﬀer and stained with
Annexin-V-FITC and PI according to the procedure from the manufac-
2.8. Growth kinetics study
turer's manual. Finally, the fluorescence intensity of the stained MCF-
7 cells was determined using CytoFLEX S flow cytometry
The growth kinetics of treated and non-treated MCF-7 breast cancer (Moghadamtousi et al., 2014).
cells with IL-GFE and Taxol as a positive control (+ve) was studied and
compared to observe the antiproliferative eﬀects of IL-GFE on the 2.11. Statistical analysis
growth of MCF-7 cell lines. Firstly, MCF-7 cells were harvested and
seeded into T-25 flasks at 2 × 105 cells/mL. There were three replicates The cell viability experiments were carried out in triplicates, and
of flasks in this study: untreated MCF-7; IL-GFE-treated MCF-7 at the each data point represents the overall mean of at least 3 independent
IC50 value of 4.75 μg/mL and taxol-treated MCF-7 at the IC50 value of experiments. The findings were expressed as mean ± standard devia-
asks to count 7.00) and Microsoft Oﬃce, Excel software to carry out the statistical
samples every 8 h interval from to 144 h. Each sample was performed
in three independent experiments. After incubation for 24 h, media was analysis. One-way analysis of variance technique followed by Tukey's
removed, and fresh media containing samples as described before with test was applied to observe the significance between the groups. The
the IC50 value concentrations were added into separate flasks. Then, entire statistical analysis was carried out at (p ≤ 0.05).