MCF-7 and MCF-7-ADR (MCF-7-adriamycin-resistant breast cancer cells) cell lines were obtained from the Culture Collection Company (ATCC-LGC Promochem, Teddington, UK). Cells were routinely grown in DMEM (Invitrogen) medium supplemented with 10% FBS (Invitrogen) and 100 units of penicillin/streptomycin per mL (Invitrogen) at 37 ℃ and 5% CO2 under humidifying conditions. The medium was refreshed every two or three days regularly until the cells reached 80-90% confluence, and the cells were transferred to next experiment or make stock solutions. The mycoplasma contamination was tested every month.
MTA (reference substance solution), which was used in quantitative analysis in a reverse phase-high performance liquid chromatography (RP-HPLC) experiment, was purchased from Sigma-Aldrich (St. Louis, MO). MTA used for cell incubation, was purchased from Eli Lilly company. ABCC5 human shRNA was bought from OriGENE company (Locus ID 10057, Product ID TL315024). Rabbit antihuman Anti-MRP5 antibody [M5II-54](ab137070, Abcam), rabbit antihuman Bax mAb (5023, CST), Rabbit antihuman caspase-3 mAb (14220, Cell Signaling Technology), cleaved caspase-3 mAb (9664, Cell Signaling Technology) and β-Actin (13E5) rabbit mAb (4970, Cell Signaling Technology) were used as the primary antibodies, and the goat anti-rabbit IgG (final dilution 1:2000; LI-COR Biosciences, Lincoln, NE, USA) was used as the secondary antibody.
Generation of ABCC5 adenovirus
For recombinant adenovirus construction, the ABCC5-gene cDNA (AdvABCC5) and the green fluorescence protein gene cDNA (AdvCtrl, control) were cloned by PCR and inserted into pHBAD-EF1-MCS-3flag-CMV-EGFP vector (supplemental Fig.1). The pDC315-ABCC5 and pBHGloxE1,3Cre were co-transfected into HEK293 cells using Lipo-FiterTM transfection reagent (QIAGEN) to generate the recombinant adenoviruses. The recombinant AdvABCC5 and AdvCtrl adenoviruses produced in the HEK293 cells were purified and the virus titer was measured by plaque assay. The stock solutions of AdvABCC5 and AdvCtrl were 1×1011 plaque formation unit (PFU)/ml, respectively.
Collection of tumor tissue specimens from patients and primary breast cancer cell isolation
A total of 34 patients with confirmed primary BC (2 cm or larger), who consecutively underwent neoadjuvant chemotherapy containing anthracyclines at the Breast Tumor Department, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, were enrolled into this study from January 2014 to December 2015. Tumor specimens were obtained by surgical excision before MTA chemotherapy. Informed consents were signed and retrieved from all patients, following a protocol approved by the Ethics Committee of Xinhua Hospital, Shanghai Jiaotong University School of Medicine.
To isolate the BC cells, at least two consecutive frozen sections were prepared for each paraffin embedded tumor tissue sample, and one of the sections was then subjected to the hematoxylin-eosin staining to confirm the presence of cancer cell, and the adjacent one was transferred to cancer cell isolation as described in the previous publication . Briefly, the blood, fat and fibro connective tissue were removed from the tumor tissue pieces, and then the residual was cut into 1-2 mm pieces for enzymatic disaggregation. The small tissue pieces were incubated with 2.5% crude trypsin for 30 minutes at 37°C and with collagenase (0.15%) overnight. Cells released after enzymatic treatments were further tested for cell viability and were cultured to perform subsequent experiments.
Twenty-four female Balc/b nude mice (5 weeks, 18 g) were purchased from the Shanghai Super B&K Laboratory Animal Corp. Ltd. (Shanghai, China), and all the mice were raised in specific pathogen-free environment with free access to food and water. All animal studies were approved by the Research Ethics Committee at Xinhua hospital, affiliated to Shanghai Jiaotong University, school of Medicine.
On day 0, every mouse was injected with 1×107 MCF-7 cells subcutaneously into the right armpit. When tumors were approximately 100 mm3 in size on day 30, adenoviruses containing ABCC5(AdvABCC5) (5×1011 PFU) were injected into the tumors of 12 mice to over-express ABCC5 in the tumor cells, and the vehicle (AdvCtrl) was applied to the other 12 control mice. The expression of ABCC5 was checked by diffused green fluorescence. When the volumes of tumors were approximately 150 mm3on day 35, 6 of the ABCC5 over-expression mice and 6 mice in the control group were treated with intravenous injections (via the tail vein) of MTA (20 mg/kg, saline) once a day, and the same dose of vehicle was administrated to the others from day 35 to 46. The tumor volumes (V) were measured using a caliper once a day (V = width2 × length/2). Mice were killed at the end of 7-week, and tumor volume and weight were measured. The animal experiments’ design has been shown in Fig.1
Measuring the MTA in MCF-7 cell using RP-HPLC
MTA concentrations in the MCF-7 cell were determinated based on a developed HPLC method. The chromatographic separation and quantification were performed on an RP-column (ZORBAX Eclipse XDB-C8, 250 mm×4.6 mm, 5 μm; Agilent) with the column temperature maintained at 25 °C, and the MTA was detected with a ultraviolet detector at a wavelength of 240 nm. The mobile phase was composed of water plus 0.02 M phosphate buffer (pH 4.0)/acetonitrile (86:14, V:V) and delivered at a flow rate of 1 mL/min. The sample pretreatment was completed using an ultrafiltration method (0.22 μm). All experiments were completed on an Agilent 1260 HPLC system. A calibration curve was constructed at a range of 80-625 ng/mL for the MTA measurement. The injection volume was 20 μL, and all analysis was performed in triplicate.
Cell preparation for cellular uptake analysis
MCF-7 cells were seeded at 2×105/well into six-well flat-bottom tissue-culture plates in triplicate. After 24 hours, the cells were infected with ABCC5, Ctrl and siABCC5-RNA expressing adenovirus, separately. The cells were incubated with adenoviral particles for another 24 hours and then refreshed with medium containing 50 μM MTA(21.37 μg/mL). At the particular time points (0, 0.5, 1, 2, 4, 24 hours), the cells were washed three times with cold PBS (0.1 M, pH 7.4) and then resuspended in 0.2 mL of RPMI-1640 medium and homogenized. After centrifugation at 13000 ×g for 10 min, the supernatant was harvested and stored at -80°C for the detection of MTA by the RP-HPLC method as mentioned above.
Cell viability assay
Cell viability assay was performed using CCK-8 kit (Dojindo Laboratories) according to the manufacturer's instructions. Briefly, cells were plated in 96-well plates at a density of 2000 cells/well, and then treated with different concentrations of MTA for 72 h. Then CCK-8 solution diluted with DMEM/F12 with 10% FBS at 1/10 ratio was added to each well and incubated for 2 hours at 37°C. Finally, the absorbance at 450 nm was measured using a microplate reader. The (%) cell viability was calculated using the formulae; (OD treatment group-ODBlank)/(OD control group-OD Blank) × 100. The IC50 value was determined using GraphPad Prism software. All experiments were performed in triplicate, and the presented data represent the mean of three biological repeats.
Protein extracts were separated through 5% to 12% SDSPAGE, and then transferred to nitrocellulose membranes, probing with mouse monoclonal antibodies against ABCC5 or β-actin, and then followed by incubation with either IRDye 700 or 800 secondary antibodies and visualized using the Odyssey Infrared Imaging System software (Li-Cor, Lincoln, NE).
RNA Isolation and Real-Time RT-PCR
Total RNA was extracted from the cells using RNeasy mini kit (QIAGEN) and qRT-PCR were performed on the cDNAs generated from 250 ng of total RNA by using HotStart-IT® SYBR® Green qPCR Master Mix along with UDG (2X) by a user-friendly TM kit (USB Corporation). ABC transporters subfamily primers were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd. (supplemental Table.1).
Cells were initially seeded onto coverslips, and then harvested and washed three times with PBS. Cells were fixed with 4% paraformaldehyde for 30 min at room temperature and blocked with 1% (w/v) bovine serum albumin, 0.1% Triton X-100, and 0.05% Tween-20 overnight at 4°C to avoid nonspecific staining. Next, the cells were incubated with goat polyclonal antibody, anti-ABCC5 for overnight. Subsequently, the secondary antibody (TRITC-rabbit-anti-goat, 1:100) was added and the cells were incubated for 1 hour in dark room. DAPI staining was used to visualize the cell nuclei. The images were captured by Leica DMI300B inverted fluorescent microscope.
The results were presented as mean ± SD. The graphics and calculations were finished using Microsoft excel software (Microsoft Corp) or Prism 5.0 software (GraphPad Software Inc). The IC50 values were calculated by nonlinear regression from a sigmoidal dose-response curve (variable slope, bottom value 0) using Prism software. Pearson’s correlation test was used to analyze the correlation between the expression of target gene and the IC50 values. The p value<0.05 was considered statistically significant using unpaired t test analysis unless stated otherwise.