2.1. Cell culture
Human breast cancer cell lines MCF7, MB231, T47D and BT549 were purchased from the American Type Culture Collection (Manassas, VA, USA). Cell lines were cultured at 37 ℃ in a humidified atmosphere of 5% CO2-95% air. T47D cells were cultured in DMEM medium with 10% fetal bovine serum (FBS), 50 units/mL streptomycin and 100 units/mL penicillin; MCF7, MB231 and BT549 cells were cultured in RPMI medium with 10% FBS, 50 units/mL streptomycin and 100 units/mL penicillin. All cell lines were tested and found to be free of mycoplasma contamination using MycoAlertb Mycoplasma Detection Kit (Lonza, Basel, Switzerland). When used for assays, cells were seeded in multi-well culture plates, and cultured in phenol red-free DMEM/RPMI-1640 medium. After 24 h, cells were washed with phosphate-buffered saline (PBS) and then treated with carbidopa (Sigma, St. Louis, MO, USA) for 24 h. CH223191 (Sigma, St. Louis, MO, USA) was used as a selective blocker of AhR. MG132 (Sigma, St. Louis, MO, USA) was used as a proteasome inhibitor.
2.2. Animals
Female athymic BALB/c-nude mice (7-week-old) were purchased from Vital River Laboratories (Beijing, China). The animals were maintained under a specific pathogen-free housing conditions in a barrier facility. They had free access to rodent chow and water. All animal studies were carried out in accordance with the Guidelines for Animal Experimentation of Wenzhou Medical University, and the Animal Ethics Committee of the institution approved the protocol.
2.3. Cell proliferation assay
Cell proliferation was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. MCF7 and MB231 cells were seeded in 96-well plates (2 × 103 cells/well). After 12 h, the cells were incubated for 24 h in the medium containing 0–30 µM carbidopa. Then, the medium was replaced with fresh medium containing MTT reagent (5 mg/mL in PBS; Solarbio, Beijing, China) and incubated for additional 4 h. The reaction product was dissolved in 150 µL of dimethyl sulfoxide and the absorbance at 490 nm quantified with a plate reader.
2.4. Colony-formation assay
For colony-formation assay, the cells were seeded into 6-well plates (2 × 103 cells/well). After 12 h, the cells were incubated in the medium containing carbidopa. The medium was changed every 3 days. When control cells reached 60–70% confluency, control cells and carbidopa-exposed cells were fixed with ice-cold 100% methanol for 20 min. The cells were then incubated with 0.1% crystal violet (Solarbio, Beijing, China) for 2 h. The cells were then washed with water and then imaged. The colonies counted under a light microscope (Leica, Wetzlar, Germany).
2.5. Cell-migration assay
Cells were seeded in six-well plates and incubated in the medium until grown to 90% confluency. A wound was created by scrapping the cells in the middle of the confluent culture with a sterile 200 µL pipette tip. Cells were cultured in the same medium for another 24 h. Then, the medium was replaced with PBS and the wound gap was photographed with an inverted microscope (Leica DM 14000B microscope fixed with digital camera, Wetzlar, Germany) at 0 h and 24 h.
2.6. Apoptosis analysis
Apoptosis was determined by flow cytometry and TUNEL staining. For flow cytometry, the cells were stained with Annexin V/FITC-propidium iodide (PI) using Apoptosis Detection kit (Beyotime Biotechnology, Shanghai, China). Tumor tissues were fixed in 10% formalin and embedded in paraffin. Specimens were sectioned at 5-µm thickness. For TUNEL assay, the MCF7 cells and tumor tissue sections were stained with an In Situ Cell Death Detection Kit (Roche, Basel, Switzerland). The stained cells were imaged with a confocal laser scanning microscope (TCS SP8, Leica, Wetzlar, Germany). The number of cells per field was counted and the percent of TUNEL-positive cells was calculated.
2.7. RNA isolation and PCR
RNA was extracted from the cells using TRIZOL Reagent (Takara, Japan). RNA (2 µg) was reverse-transcribed into cDNA (GoScript Reverse Transcription Kit; Promega, Madison, WI, USA). RT-PCR was conducted in a Thermal Cycler (T100, Bio-Rad, Hercules, CA, USA). PCR products were electrophoresed in agarose gel and visualized with ethidium bromide (GelDoc XR+; Bio-Rad, Hercules, CA, USA). Quantitative PCR (qPCR) was conducted using FastSYBR Green qPCR Master Mix (Takara, Japan) and Viia7 real time PCR machine (Bio-Rad). Changes in expression were calculated as the ratio of expression in treated samples to expression in control samples. The expression levels for target genes were normalized to the expression of the GAPDH (glyceraldehyde-3-phosphate dehydrogenase). PCR primers were as follows: ERα: 5'-TTCTTCATTTCCCAGCGT-3' (sense) and 5'-CTAAACCACCAAGGGCATA-3' (antisense); cyclin D1: 5'-CCGTCCATGCGGAAGATC-3' (sense), 5'-ATGGCCAGCGGGAAGAC-3' (antisense); c-Myc: 5'-GCCAGAGGAGGAACGAG-3' (sense), 5'-GCTTGGACGGACAGGAT-3' (antisense); GAPDH: 5'-CGGAGTCAACGGATTTGGTCGTAT-3' (sense), 5'-AGCCTTCTCCATGGTGGTGAAGA-3' (antisense); CYP1B1: 5'-GGGCTACCACATTCCCA-3' (sense), 5'-GAGGCCATCCTTGTCCA-3' (antisense); ARNT: 5'-GGTTTGGCAGCACACTCTATG-3' (sense), 5'-ACAGTTATCCTGGCCTCCGT-3' (antisense); CYP1A1: 5'-CAAGGGGCGTTGTGTCTTTG-3' (sense), 5’-GTCGATAGCACCATCAGGGG-3' (antisense).
2.8. Western blot
Lysates from cells and tumor tissues were prepared and protein determined using the BCA assay (Beyotime Biotechnology). Protein (30 µg) from each sample was resolved on SDS-PAGE, and transferred to PVDF membrane. Membranes were blocked with 5% bovine serum albumin in Tris-buffered saline containing 0.1% Tween 20 and incubated with primary antibodies overnight at 4°C. Membranes were washed thrice, 5 min each time, and then incubated with either horseradish peroxidase (HRP)-conjugated goat anti-mouse (ab6789, Abcam, Cambridge, UK) or HRP-conjugated goat anti-rabbit (ab6721, Abcam) secondary antibodies for 2 h at room temperature. Immunoreactive bands were visualized using Pierce ECL plus Western blotting substrate (Thermo Fisher Scientific, Waltham, MA, USA). The primary antibodies used were: ERα (sc-8002, Santa Cruz Biotechnology, Dallas, TX, USA), HSP90 (ab203126, Abcam), β-actin (4970S, Cell Signaling Technology, Boston, MA, USA), Bax (2774S, Cell Signaling Technology), Bcl-2 (15071S, Cell Signaling Technology), ubiquitin (3936S, Cell Signaling Technology), CYP1A1 (ab124295, Abcam) and AhR (ab190797, Abcam). The protein bands were analyzed using ImageQuant 5.2 software. β-Actin was used as loading control.
2.9. Immunofluorescence staining
Immunofluorescence staining was performed for nuclear localization of AhR in MCF7 cells. Cells in chamber slides were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.5% Triton-X 100 (Solabio, Beijing, China) for 10 min at 37°C. After washing, the cells were blocked in 5% BSA for 1 h and incubated with primary rabbit antibody against AhR at 4°C overnight. Cells were then washed and incubated with Alexa Fluor 488-conjugated anti-mouse IgG secondary antibody (ab150113, Abcam) for 1 h. After subsequent washing with PBS, the cell nuclei were stained with DAPI for 15 min. Photo capture was performed by the confocal laser scanning microscope (TCS SP8, Leica).
2.10. Luciferase reporter assay
MCF7 cells were transfected with AhR-luciferase reporter plasmid (YEASEN, Shanghai, China) and pRL (renilla luciferase)-null plasmid (internal control) using Lipofectamine 2000. After 24 h, cells were serum-starved and treated with or without carbidopa. Cell lysates were used to measure luciferase activity using the Dual Luciferase Reporter Assay System (E1910, Promega, Madison, WI, USA). Firefly luciferase and renilla luciferase were detected on a Veritas Microplate Luminometer (Promega). Normalization was done with renilla luciferase.
2.11. Immunoprecipitation
MCF7 cells were lysed with IGEPAL CA-630 buffer: 50 mM Tris-HCl, pH 7.4, (Sigma, T5030), 1% IGEPAL CA-630 (Sigma, I8896), 10 mM EDTA, 150 mM NaCl, 50 mM NaF, 1 µM leupeptin (Sigma, L5793), 0.1 µM aprotinin (Sigma, SRE0050). Primary antibody was covalently immobilized on protein A/G agarose using the Pierce Crosslink Immunoprecipitation Kit (Thermo Scientific, 26147). Samples were incubated with immobilized antibody beads for at least 2 h at 4°C. Cell lysates were also subjected to immunoprecipitation with either mouse IgG isotype control (Cell Signaling Technology, 5415) or rabbit IgG isotype control (Cell Signaling Technology, 3900) according to the immunoglobulin type of the primary antibody. After immunoprecipitation, the samples were washed with PBS for 5 times. They were then eluted with glycine-HCl (0.1 M, pH 3.5) and the immunoprecipitates were then subjected to immunoblotting using specific primary antibodies described in the Western blot part.
2.12. Molecule docking
As there is no information available in the literature on the structure of the ligand-binding domain of AhR, a homology model has been developed for the PAS-B domain in AhR that interacts with agonists for the purpose of molecular modeling and docking simulations [30, 31]. With this model as the template, we analyzed the interaction of carbidopa and the endogenous AhR agonist L-kynurenine with the PAS-B domain of human AhR based on AutoDock Vina [32]. The size of the grid box was set to 12 Å × 12 Å × 12 Å. The exhaustiveness value was set to 8.
2.13. Xenograft studies in athymic BALB/c nude mice
BALB/c nude mice were randomly divided into two groups. The investigators performing the outcome analyses were blinded to the group assignment. MCF7 cells were subcutaneously injected into the right flank of each mouse (0.1 mL, 1 × 106 cells per mouse). When tumors reached a volume of 100 mm3, mice were randomized into control and treatment groups (6 mice per group); no specific criteria or method was used for randomization. The treatment group received intraperitoneal (i.p.) injection of carbidopa (7.5 mg/kg; 0.1 mL volume) and the control group the same amount of vehicle twice a day, every 12 h. The treatment continued for 14 days continuously. The body weight and tumor volume were recorded every two days throughout the procedure. Tumor volumes were determined by measuring length (L) and width (W) of the tumors periodically and then calculating the volume using the formula: V = 0.5 × L × W2 [33]. After 14 days, mice were euthanized under anesthesia. Tumor specimens were harvested, weighed, and then processed for histology and protein assays.
2.14. Immunohistochemistry
Tumor tissues were fixed in 10% formalin and embedded in paraffin. Specimens were sectioned at 5-µm thickness. The sections were stained using routine immunohistochemical techniques. Briefly, after pre-treatment with heat-mediated antigen retrieval for 20 min in a microwave oven, the endogenous peroxidase activity was blocked by incubating the tissues in 3% H2O2 for 30 min. After blocking for 15 min with 10% normal goat serum, the sections were treated with primary antibodies against Ki67 (1:500 dilution; overnight exposure) (ab15580, Abcam). HRP-conjugated secondary antibodies were used for detection. Next, the sections were exposed to Diaminobenzidine (DAB) solution (P0203, Beyotime Biotechnology), and counterstained by Hematoxylin Staining Solution (C0107, Beyotime Biotechnology). Finally, tumor specimens were observed under the microscope. The substitution of PBS for primary antibody was used as negative control. For Ki-67 index, numbers of positively stained cells were expressed as a percentage of the total number of cells examined.
2.15. Statistical analysis
Results are expressed as means ± SD. Statistical differences were assessed with the unpaired two-tailed Student’s t-test for two groups and with one-way ANOVA for multiple groups using SPSS software. Bonferroni’s post-hoc testing was employed after ANOVA to test for significant differences between groups. p < 0.05 was considered statistically significant. Statistical analyses were done using GraphPad Prism (GraphPad Software).