Cell culture
The breast cancer cell lines MCF7 and MDA-MB-231 were derived from ATCC. Breast cancer cell lines were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Invitrogen, CA, USA). All cells were cultured at 37 ℃ and 5% CO2.
MTT assay
MTT method was used to determine cell viability of tumor cells after lidocaine treatment. In short, the cells (5–6×103) per well were seeded on a 96-well plate and cultured for 24 hours, and then exposed to lidocaine at 0mM, 3mM, 6mM for 12h, 24h, 48h. 20 µl 5mg/ml MTT was added to each well and incubated for another 4 h at 37°C. The supernatant was then removed.100µl DMSO was added to each well and gently rocked incubated for 10 minutes. The absorbance (OD) was measured at 490nm by SpectraMax iD3 ( Molecular Devices).
Cell proliferation assay
Cells were seeded on 24-well plates at 3000 cells per well in 0.5 ml DMEM with 10% FBS. Normally, the culture medium was changed every two days. 24 hours before cell treatment, the medium was replaced with DMEM supplemented with lidocaine at indicated concentrations. At indicated time points, cells were fixed in 4% formaldehyde and stained with 2% crystal violet. Dye was extracted with 10% acetic acid and the relative proliferation was determined by the absorbance at 595nm(SpectraMax iD3,Molecular Devices).
Protein Isolation And Western Blot
Protein isolation and Western blot
Protein extracts were prepared using NP-40 lysis buffer containing phosphatase and protease inhibitors. The cell lysates were then subjected to SDS-PAGE followed by immunoblot using indicated antibodies(CDK1, Proteintech, No.19532-1-AP, 1:2000;cyclinB1, Proteintech,No. 55004-1-AP,1:1000;BCL2, Proteintech,No. 12789-1-AP,1:2000;BCL-XL, Proteintech,No. 26967-1-AP,1:1000;p62, Proteintech, No. 18420-1-AP,1:2000;LC3B, Proteintech,No. 14600-1-AP,1:1000;p-ERK, Proteintech,No. 24390-1-AP,1:1000;ERK, Proteintech,No. 16443-1-AP, 1:2000; p-AKT, Proteintech,No. 66444-1-Ig,1:5000;AKT, Proteintech,No. 10176-2-AP,1:2000;β-actin, Proteintech,No. 66009-1-Ig,1:5000).
Flow cytometry for cell cycle analysis
Before treated with lidocaine, all breast cell lines were synchronized by serum starvation for 24 hours. Cells treated with lidocaine were digested with trypsin. After centrifugation, the supernatants were discarded, and the cell precipitations were resuspended in 5 ml PBS, then centrifuged again and resuspended in 0.5 ml PBS. The cells were then fixed on ice with 70% alcohol for at least 2 hours, and then centrifuged at 1500 RPM (Revolutions Per minute) for 10 minutes. The supernatants were discarded and cell precipitations were then resuspended with PBS and centrifuged again. Cell precipitations were resuspended with 500 µl guava cell cycle reagent. After incubation at 37 ℃ for 30 min in the dark, cell cycle distribution was analyzed by the BD FACSMelody™ flow cytometer for counting 30000 cells.
Flow cytometry for apoptosis analysis
The lidocaine-treated cells were digested with trypsin, centrifuged at 300g for 5 minutes, the supernatants were discarded, the cells were collected, washed again with PBS, and then gently resuspended and counted. 5 × 105 cells were collected and centrifuged at 300g for 5 minutes, and the supernatants were discarded. The cells were resuspended with PBS again. After centrifugation, the supernatants were discarded. The cells were resuspended with 100 ul diluted 1×Annexin V Binding Buffer. 2.5 ul Annexin V-FITC and 2.5 ul propidium iodide (PI) staining were then added. The samples were incubated for 15 minutes in the dark at room temperature. 400ul diluted 1×Annexin V Binding Buffer were then added to mix the samples, apoptosis was analyzed by flow cytometer (BD FACSMelodyTM).
Quantitative RT-PCR
Total RNA was extracted using Trizol reagent (Invitrogen) and 1 µg total RNA was performed reverse transcription using PrimeScript RT reagent kit with gDNA eraser (TaKaRa), according to the manufacturer’s instructions. Quantitative RT-PCR was performed with SYBR Green dye using (Applied Biosystems). The relative amount of cDNA was calculated by the comparative Ct method using GAPDH as a control. PCR reactions were performed in triplicate.The specific primer sequences were presented as follows:CDK1:F 5ʹ-GGAAGGGGTTCCTAGTACTGC-3ʹ,R 5ʹ-CCATGTACTGACCAGGAGGG-3ʹ;cyclinB1:F 5ʹ-GCACTTCCTTCGGAGAGCAT-3ʹ, R 5ʹ-TGTTCTTGACAGTCCATTCACCA-3ʹ; GAPDH:F 5'-GCACCGTCAAGGCTGAGAAC-3',R 5'-TGGTGAAGACGCCAGTGGA-3'.
Statistical analysis
The statistical analysis was performed with SPSS software, version 19.0(SPSS, INC., USA). All the experiments in vitro were performed a minimum of three times. Data were expressed as the mean ± standard deviation (SD). The results were demonstrated by charts using GraphPad Prism version 7.0(GraphPad Software, Inc., USA). The data were compared using a one-way ANOVA followed by a Dunnett post hoc test, as appropriate; When variance between groups was not homogeneous and they did not show normal distribution, data were compared using non-parametric Kruskal-Wallis rank sum test. Differences was considered statistically significant when P < 0.05.