In-silico analysis
The cBioPortal for Cancer Genomics database is an open-access resource at http://www.cbioportal.org used for genomic alteration, visualization, analysis and correlation of the mRNA levels between individual genes (CTNNB1, SANI1, ZEB1, VIM and CDH) and Pearson correlation analysis was used to attain their correlation coefficients.
The bcGenExMiner v4.1 database at http://bcgenex.ico.unicancer.fr/ was used to evaluate the relationship among the expression of β-catenin, E-Cadherin, Vimentin, SNAIL and ZEB1.
The Kaplan Meier Plotter (http://kmplot.com/analysis/) used for evaluating the biological relationships between survival information and gene expression levels. This tool provides powerful platform prognostic values of each selected genes.
Cell culture
Human breast cancer cell line (MDA-MB-231) were purchased from the Pasteur Institute Cell Bank of Iran. Cells were cultured and maintained in RPMI1640 medium containing 10% FBS and 1% penicillin/streptomycin. Cells were incubated in a humidified atmosphere of 5% CO2 at 37 ◦C. All assays were done using cells in exponential cell growth phase. Twenty-four hours after seeding, the cells were treated with culture medium containing different concentrations of crocin.
Cell cytotoxicity assay
In-vitro, cell proliferation and cytotoxicity were monitored by standard MTT assay for treated and untreated control MDA-MB-231 cell line. MTT experiment is a colorimetric method with highly accuracy and broadly used to determine cell viability and cytotoxicity, chiefly in the development of novel therapeutics.
MDA-MB-231 breast cancer cells were harvested at exponential growth phase with 0.05% trypsin-EDTA and seeded in 96-well plates (SPL, Korea) at a density of 5,000 cells per well. After 24h incubation, cells were treated with 0, 5, 10, 20,40, 80, 160, 320, and 640 µM concentration of crocin. After 24,48 and 72 h of incubation period, the supernatant culture medium was eliminated and MTT solution (0.5 mg/ml, 50 µl) (Sigma, Germany) were added and cells were incubated for another 4 h. Then, 200 μL of DMSO (dimethyl sulfoxide) was added to each well for to dissolve formazan crystals and the absorbance of each well was obtained using a 3200 statfax ELISA plate reader (statfax, USA) at 570 nm (with a reference wavelength of 650 nm). The IC50 value (Concentrations that reduce the cell population up to 50 %) were calculated by means of GraphPad Prism 6.01 software (GraphPadSoftware Inc., USA).
Cell migration assay
CytoSelect TM 24-well cell migration and invasion assay utilize for Cell trans-well migration assay (8-µm pore size; colorimetric format; Cell Biolabs, Inc., USA) this test applied for assessment of crocin effect on the migration and invasion of the MDA-MB-231 cells. Assay was performed According to the manufacturer’s protocol. In brief, cells treating with 60, 120 and 240 µM of crocin for 48 h. After treatment period, a 500,000 cells/ml cell suspension was prepared and transferred into the upper chamber of the plate. 150 µl complete RPMI1640 medium containing 10 % FBS as chemoattractant loaded to the bottom chamber and plate were placed for 24 h in humified incubator. In response to chemoattractant, migratory cells passed through polycarbonate membrane pores and invaded to the bottom of the membrane. Lastly, migrated cells were stained, extracted, and quantified at A560 nm as pronounced in the manufacturer protocol.
Wound healing assay
wound healing assay were used for assess crocin effect on migration of MDA-MB-231 cells. Six-well plate seeded with 5 × 105 cells in fresh medium and incubated in humified incubator to forming the monolayer with 100 % confluency. Subsequently, in each replicate well of monolayer cells a straight scratch was generated using a sterile yellow pipette tip. To remove detached cells and debris, plates were washed twice with PBS. Then plates incubated with fresh RPMI1640 complete medium in the absence or presence of crocin for 48 h (Grada et al., 2017).
The width of the wound was monitored by inverted microscope (Olympos, Japan) within a desired time frame and photographed. ImageJ software (National institute of Health, Bethesda, MD, USA) were used for measurement of area of wound. Relative area of wound was calculated from each triplicate treatment and the data were presented as mean ± SD.
Quantitative Real-Time PCR
After 48 h of crocin treatment, desired gene expressions were assessed by Real-time polymerase chain reaction. Extraction of total RNA was performed using RNX-plus reagent (SinaClon, Iran) according to the manufacturer’s protocol. cDNA was synthesized using Pars toos RT Reagent kit with 1 µg total RNA (Pars toos, Iran). qRT‐PCR was performed in three replicates of each sample with specific primers for β-catenin, E-cadherin, Vimentin, Snail and Zeb-1(Table 1) in a 20 μL reaction mixture microtube containing, 1 μL of 0.5 mM of primer, 10 μL SYBR Green master mix, 5 μL DW and 4 μL of cDNA in PCR micro tube. Rotor gene 6000 system (corrbet, Australia) were used for amplification reaction. Expression level fold change of each mRNA sample normalized against the β-actin mRNA and quantified based on of the comparative 2-ΔΔCt method.
Statistical analysis
Statistical analyses performed by GraphPadPrism 6.01 software. Results were demonstrated as the mean ± standard deviation (SD). Unpaired student t-test for assessment of statistical differences were used; and P value less than 0.05 was considered significant.