Cell culture
MDA-MB-231, MCF-7 (breast carcinoma cancer) cell lines were obtained from the National Cell Bank of Iran (NCBI) affiliated to the Pasteur Institute of Iran (Tehran, Iran). The cells were cultured in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco) 100 IU/ml penicillin and 100 mg/ml streptomycin (Gibco) under a humidified atmosphere (95 %) at 37 °C with 5 % CO2. All the cells used in the current experiment were at passage 3–6. Melatonin (Sigma-Aldrich) was dissolved in dimethyl sulfoxide (DMSO, Merck, Germany) to prepare 1 M as our stock solution. After that, the stock solution was diluted with DMEM/LG (Gibco, USA) to achieve a final concentration of 0.1 mM as an effective concentration. The maximum concentration of DMSO for each experimental condition was less than 0.01%.
Cell viability assay
MTT assay was applied to evaluate IC50 of Melatonin. First, the cells (104 cells/well) were plated in 96-well culture plates. After incubation of the cells with different concentrations of melatonin, the media was replaced with fresh media containing 2 mg/ml MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide) (Roche Molecular Biochemicals, Indianapolis, IN) solution and incubated for an additional four h at 37 °C. Then, the solution was replaced with a 200µl DMSO solution. Finally, the absorbance value was measured at 570 nm using a Micro-plate reader system (BioTek, USA). IC50 was determined for each agent by calculating the slope and intercept of different concentrations. The experiment was performed in triplicate and repeated three times. After determining the IC50 values for melatonin, all experiments were performed in 6 groups, including control groups of MCF-7 and MDA-MB-231 cell lines and melatonin treated groups(0.625mM) absence or presence of EGF (100 ng/mL) for 48h.
Flow cytometry analysis
According to the manufacturer's instructions, a flow cytometry assay was performed to evaluate cancer stem cells' surface markers. Briefly, 24 h post-treatment, cells were washed twice with PBS solution and diluted into 100µl of flow cytometry buffer (PBS solution/BSA 0.5%, pH 7.2). Then the suspension was incubated with 10µl of both PE-CD44 (Miltenyi Biotec, Germany) and FITC-CD24 (Miltenyi Biotec, Germany) antibodies for 10 minutes at 4°C. Finally, the cells were centrifuged and immersed in the buffer to remove the unconjugated antibodies. Samples were analyzed by the FACSCalibur™ (BD Bioscience, NJ, USA) and processed by FlowJo ver. 7.6.1 software. Appropriate isotype-matched antibodies identified nonspecific protein labeling.
Cell proliferation assay
After harvesting and counting 48-hour-treated cells, 100µl flow cytometry buffer (PBS solution supplemented with 0.5% BSA, pH 7.2) was added into the cell pellet (1×106 cells). The cells were treated with 0.1% Triton X100 for 1 minute to make the cell membrane relatively permeable. After centrifugation (at 300×g for 10 minutes), the supernatant was discarded, and the cell pellet was incubated (at 4°C for 20 minutes) in 100 µl flow cytometry buffer containing 5µl anti-Ki-67 FITC antibody (Miltenyi Biotec, Germany). Then, 1-2 ml of buffer was added to the suspension, and the unconjugated antibodies were removed through centrifugation. Finally, the cell pellet was resuspended into the 500µl buffer and proceeded to flow cytometry analysis. Samples were analyzed by the FACSCalibur™ (BD Bioscience, NJ, USA) and processed by FlowJo ver. 7.6.1 software.
Cell apoptosis assay
According to the manufacturer's protocol, the cell's apoptosis rate is determined by a commercial Annexin V/PI Apoptosis Detection Assay kit (EXBIO). Briefly, After 24h incubation with the desired concentration of melatonin in the presence and absence of EGF, the cells were cultured. They washed with PBS, stained with Annexin V–FITC/PI, and analyzed for apoptotic cells using FACSCalibur™ (BD Bioscience, NJ, USA) FlowJo (7.6.1) software for acquisition and analysis as described before.
RNA isolation and real-time RT PCR
According to the manufacturer's procedure, to analyze the expression level of genes involved in EMT and invasion, total RNA was extracted using Total RNA isolation solution RiboEx (GeneAll). The extracted RNA was reverse-transcribed into cDNA by GeneAll Kit, and Real-time RT PCR performed using particular primers (listed in Table 1). Real-time RT PCR was performed using QuantiTect SYRB Green dye (TaKaRa, Japan) and Rosch system. The expression levels of each gene were analyzed by Pfaffl methods with normalization to the housekeeping gene, Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH). All experiments were carried out in triplicate.
Table 1: Sequences of primers used for real-time PCR analysis.
Reverse primer
|
Forward primer
|
Gene
|
5´-CTGTCTTCATCCTCTTCCCTTGT
|
5´-CTGGAGAAAAGCCCTATCAATGT
|
ZEB1
|
5´-CCCGTGTGTAGCCATAAGA
|
5´-CAGCCATTACCCAGTTAAGA
|
ZEB2
|
5´-CAGAGTCCCAGATGAGCATT
|
5´-GAGTTTACCTTCCAGCAGCC
|
Snail
|
5´-TAGGTGGCAATCTCAATGTCAA
|
5´-AGATGCGTGAAATGGAAGAGAA
|
Vimentin
|
5´-GTGTATGTGGCAATGCGTTC
|
5´-TGCCCAGAAAATGAAAAAGG
|
E-cadherin
|
5´-CCCATCACGCCACAGTTTCC
|
5´-CAAGATCATCACCAATGCCT
|
Β-actin
|
5´-CGGCCACTCAGTAGGTGTCTTT
|
5´-CACATAGTGATGGTTCCCCTGTT
|
MMP2
|
5´-ATCCGGCAAACTGGCTCCTTC
|
5´-ATTTCTGCCAGGACCGCTTCTAC
|
MMP9
|
Western blot analysis
The western blot analysis was performed as previously described (9). Cells (1.5×106) lysed in a protein extraction buffer (25 mm HEPES, 1% Triton X-100, 2 mm EDTA, 0.1 m NaCl,25 mm NaF, 1 mm Sodium Orthovanadate) containing protease cocktail inhibitor (Roche, Basel, Switzerland) for 30 min on ice. Cell lysates were centrifuged at 12000 g for 20 min. The supernatant was then collected, and concentration of protein was determined with picodrop (Picodrop LTD, Cambridge, UK). The equal amount of protein (~100 µg) loaded at 12% SDS- Acrylamide gel followed by transferring to the PVDF membrane. Skim milk (5%) in PBS was used to block the membrane and then incubated with phospho ERK1/2, phospho P38, ERK1/2, P38, and β-actin antibodies (Santa Cruz Biotechnology, Santa Cruz, USA) overnight at four °C. The HRP-conjugated antibody was applied as a secondary antibody (Santa Cruz Biotechnology, Santa Cruz, USA) for 1 hour at room temperature. Finally, protein detection was performed with an ECL reagent. The density of each band was determined using ImageJ software.
Transwell invasion assay
The effect of melatonin on the invasion of EGF-stimulated MCF-7 and MDA-MB-231 cells evaluated using trans-well chambers. The chamber's upper surface was coated with 30-50μL of matrigel and then incubated at 37 °C for 15-30 minutes. MCF-7 and MDA-MB-231 cells (1 × 106 cells/ml) were seeded in serum-free RPMI 1640 in the chambers and exposed to EGF and melatonin treatment (600 µM). After 24 h of incubation at 37 °C, the cells on the upper surface of the chamber were removed, and the invasive cells on the other side of the chamber were fixed with ethanol 70%. The fixed cells were stained with 0.1% crystal violet and counted using an inverted microscope (Olympus IX71, Japan) at 20 ×magnification from at least five random fields. The relative cell invasion calculated using the following equation:
Statistical analysis
All experiments were performed in triplicate. Results were expressed as mean ± standard deviation (SD) and analyzed by one-way analysis of variance (ANOVA) or two-way ANOVA using GraphPad Prism (ver: 7.0). P <0.05 was considered statistically significant.