Cell culture and treatments
The TNBC cell line MDA-MB-231 was kindly donated by Professor Erwei Song, University of Sun Yat-Sen in 11/2018. The TNBC cell lines MDA-MB-468 and HCC1937 were purchased from GeneChem Company (Shanghai, China) in 01/2019. All cells were free of mycoplasma contamination, and their identify was authenticated by short tandem repeat (STR) DNA profiling by Shanghai Biowing Applied Biotechnology Co., Ltd on 31/10/2019. All cells used in the experiments were within 30 passages after thawing. MDA-MB-231 cells were cultured in DMEM with 10% FBS (Gibco, Grand Island, NY, USA). MDA-MB-468 cells were cultured in RPMI-1640 medium with 10% FBS (Gibco). Cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. In vitro, cells were treated with 500 nM PD (PD-0332991, SelleckChem, Houston, TX, USA) or vehicle (PBS, BOSTER, Wuhan, China). MDA-MB-231 cells were treated with 50 μM CDDP, and MDA-MB-468 cells were treated with 1 μM CDDP (SelleckChem).
Apoptosis analysis
Cells (5×104/well) were seeded in triplicate in 10% RPMI-1640 medium/DMEM-FBS (complete medium) in 6-well plates and treated with PD and CDDP at the indicated concentrations separately or combined. After being treated for the defined duration, the cells were washed, resuspended in binding buffer, and stained with Annexin V-FITC/PI according to the manufacturer’s instructions (BD Biosciences). The apoptotic cell populations were analysed using flow cytometry (Beckman Coulter, CA). All assays were independently performed three times.
Cell cycle analysis
For cell cycle analysis, cells were harvested, washed with PBS, fixed in pre-chilled 70% ethanol, and kept overnight at -20°C. The fixed cells were then collected, washed, and resuspended in PBS. The cells were incubated with 1 mg/mL RNase and 50 µg/mL propidium iodide (PI) in the dark for 30 min at 37°C and subjected to flow cytometry (Beckman Coulter, CA). The cell cycle results were analysed using FlowJo version 7.6.1. All assays were independently performed in triplicate.
Assessment of cell viability
The viability of the cells was assessed with Cell Counting Kit-8 reagent (CCK8, Dojindo, Tokyo, Japan). A total of 5000 to 10000 cells per well, depending on the growth characteristics of each cell line were seeded in 96-well plates in triplicate. After adhering overnight, the designated drugs were added at different concentrations and/or sequences to the wells. After the defined duration, the supernatants were removed, and 100 μl of CCK8 solution (1:10 dilution) was added to the cells. After 2 h of incubation at 37°C in the dark, the optical density (OD) at 450 nm was measured with a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). Each experiment was performed three times. The half maximal inhibitory concentration (IC50) was determined from dose-response curves generated by GraphPad Prism version 6.0. The combination index (CI) values for the combined treatment regimens with PD and CDDP were calculated with CompuSyn version 1.0 [21].
Colony formation assay
Cells (500-1000/well) were seeded in 6-well plates and treated with the indicated drugs. After combination treatments, the medium was replenished every 3 days until cells in the control wells reached 80-100% confluence. The cell monolayers were then fixed and stained with a solution of 4% paraformaldehyde and 0.5% crystal violet for 30 min at room temperature, washed with water, and dried. Colonies containing 50 or more cells were visually identified and counted. Assays were performed with three independently treated cell populations.
Tumour xenograft studies
This study was approved by the Ethics Committees of Tongji Hospital and performed in accordance with the Guide for the Care and Treatment of Laboratory Animals of Tongji Hospital. Four-week-old female BALB/c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and quarantined alone for one week before the experiment. For MDA-MB-231 cell line xenograft models, 7×106 cells were suspended in 100 μl of PBS plus 50 μl of Matrigel (BD Biosciences, MA, USA) and subcutaneously injected into the left axilla. One week later, mice bearing engrafted tumours of 50 mm3 were randomized to receive oral treatment with 150 mg/kg PD (n=4), the intraperitoneal injection of 5 mg/kg CDDP (n=4), PD-CDDP treatment (n=4) or vehicle (PBS) treatment (n=4) according to the dosing schedule provided in Fig. 4a. The perpendicular tumour diameters were measured with callipers. Tumour volumes were calculated as (length × width2)/2 every three days. Tumours were weighed when the mice were euthanized by cervical dislocation. All mice were sacrificed when the tumour burden of the vehicle group was equal to 1000 mm3. Tumours were fixed in 4% paraformaldehyde for paraffin embedding and used for immunohistochemical staining.
Immunohistochemistry
Tissue sections were incubated with antibody against Ki-67 (#9027, 1:400) (Cell Signaling Technology) overnight at 4°C and stained with 3,3’-diaminobenzidine (DAB). Densitometry analysis was performed using ImageJ version 1.48v.
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from cells using TRIzol reagent (Invitrogen, California, USA). RB expression was measured in triplicate using SYBR Green qPCR Mix (Toyobo, Shanghai, China) according to the manufacturer’s instructions. Primer sequences were as follows: RB Forward: 5’-CTCTCGTCAGGCTTGAGTTTG-3’, RB Reverse: 5’-GACATCTCATCTAGGTCAACTGC-3’; GAPDH Forward: 5’-GGAGCGAGATCCCTCCAAAAT-3’, and GAPDH Reverse: 5’-GGCTGTTGTCATACTTCTCATGG-3’. The comparative Ct method was used to calculate the relative mRNA expression, and GAPDH was used as an internal control.
Cell transfection
Plasmids containing small hairpin RNA (shRNA) targeting RB and negative control shRNA (shNC), RB-overexpressing plasmid and empty plasmid were purchased from RiboBio (Guangzhou, China). ShRNA was transfected into MDA-MB-231 cells, and RB-overexpressing plasmid and empty plasmid were transfected into MDA-MB-468 cells with X-tremeGENE HP DNA transfection reagent (Roche, CHE) according to the manufacturer’s instructions. The shRNA sequences were as follows: shNC, 5’-TTCTCCGAACGTGTCACGT-3’, shRB, 5’-CGGCTAAATACACTTTGTGAA -3’.
Immunofluorescence assay
Breast cancer cells were subjected to indirect immunofluorescence staining with γH2AX (Ser139, #9718, 1:400) and then labelled with FITC goat anti-rabbit IgG (#AS007, 1:200). Nuclei were stained with DAPI (Life Technology). Fluorescence images were acquired using an inverted fluorescence microscope (Olympus). ImageJ version 1.48v was utilized for foci measurement and image analysis.
Western blot analysis
Cell lysates were separated by SDS-PAGE, and the proteins were then transferred to PVDF membranes. The proteins were detected using antibodies against the following: RB (ab181616, 1:2000), cyclin D1 (ab40754, 1:1000), E2F1 (ab179445, 1:1000) (Abcam, Cambridge, UK), phospho-RB (p-RB) (S780) (#8180, 1:1000), PARP/cleaved PARP (#9542, 1:1000) (Cell Signaling Technology), and β-Actin (AC026, 1:100000) (ABclonal, Boston, USA). Specific bands were visualized by ECL (Advansta, USA) and detected with an imaging system (Bio-Rad, USA).
Statistical analysis
Statistical significance was determined and means and standard deviations were calculated by GraphPad Prism version 6.0. All analyses were performed in triplicate, and P <0.05 was used to indicate statistical significance. Data are expressed as the mean ± SD or mean. The significance of differences between two groups was analysed by two-tailed Student’s t-test. The Chou-Talalay method was performed to calculate the CI.