This experimental in vitro study has been performed in applied physiology research center, in Isfahan university of Medical sciences. Human MCF7breast carcinoma has been used in our study.
2.1. Cell Culture
human MCF7 cells were obtained from Pasture institute of Iran and were grown as monolayer culture in Dulbecco’s modified Eagle’s medium (DMEM, USA) supplemented with 10% fetal bovine serum (FBS, USA), penicillin (100 U/mL), and streptomycin (100 μg/mL). All cells were incubated at 37°C in 5% CO2, under humidified air with 5% at 37°C. When the confluence was 70% - 80% (every two or three days) the cells were passed using 0.25% Trypsin- 1 µM EDTA solution followed by centrifugation (4 minutes, 1500 rpm) and replaced in T75 culture flasks at a sub-cultivation ratio of 1:10 to ensure optimal proliferation.
2.2. Drug Preparation
Duloxetine and paclitaxel were dissolved in pure dimethyl sulfoxide (DMSO). The studied concentrations were prepared in DMSO in final volumes that represent less than 1% of the total medium volume.
2.3. MTT Test
Cell viability was evaluated by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay. For this purpose, MCF7 cells were seeded on 96-well culture plates at 1 × 104 cells/well and treated with increasing concentrations of duloxetine alone ranging from 2, 20 and 100 µM and paclitaxel along with 100 nM concentration for 24 hours at 37°C. Furthermore, in order to assess the synergistic effect of paclitaxel and duloxetine, the cells were treated with both drugs concomitantly. At the time of assay, cells were washed with PBS and incubated with 10 μL of 0.5 mg/mL MTT reagent in medium for 3 hours at 37°C. Formazan crystals formed by living cells were then dissolved in 50μl of DMSO per well. The optical density (OD570-650) was then read by Multi-Detection µplate reader (BioTek Instruments, Inc.; Winooski, VT).
2.4. Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR)
Total RNA was extracted from breast cancer cells by using RNA Mini plus Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocols and cDNA was synthesized by using Revert Aid TM Reverse Transcriptase (Fermentas, Vilnius, Lithuania) with oligo-dT primers.
The primers were designed by beacon designer version 8.0:
Tau forward primer: CAACAGCGGAAGATGTGACA,
Tau reverse primer: GGACCACCTTACCACTTTCAG,
Quantitative real time RT-PCR was performed by using specific primers for tau targets and GAPDH as an internal house keeping control with the SYBR Green qPCR Master Mix (Fermentas, Vilnius, Lithuania) and run in the Rotor-gene 6000 (Corbett Life Science, Sydney Australia ). The PCR cycling conditions for the genes consisted of an initial denaturation at 95°C for 10 minutes, followed by 45 amplification cycles including denaturation at 95°C for 15 seconds, annealing at 60°C for 30 seconds and an extension at 72°C for 30 seconds.The amplification efficiencies were tested for the genes of interest, tau, and housekeeping gene. These measurements were then plotted against cycle numbers. The parameter threshold cycle (Ct) defined as the cycle number at which the first detectable fluorescence increased above the threshold observed For fold-changes calculation in relative gene expression, equation ΔΔCT, where ΔCt = Ct (Target Gene) - Ct (GAPDH) for each treatment was used.
2.5. Cell Migration Assay
For the migration assay, a total of 3 × 104 cells were seeded in the top chamber of the transwell with a noncoated polycarbonate membrane (6.5 mm diameter insert, 8.0 μm pore size). DMEM medium with 10% FBS was added to the lower chamber as a chemo attractant. After incubation for 24 hours, Cells that did not migrate through the pores were mechanically removed by a cotton swab; whereas the Cells that migrated to the lower side were detected by crystal violet staining (mean ± SEM, n = 3).
2.6. Data Analysis
Data are reported as the mean ± standard error of the mean (SEM) and followed normal distribution of independent experiments, which were done at least three times, each time with at least three independent observations. Statistical analysis was performed using one sample t-test and one-way ANOVA test. P values less than 0.05 were considered significant. Prism 6 software was used to analyzing data.
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