Materials
SiRNA-CYR61 and negative SiRNA-control were purchased from Sangon Biotech (Shanghai). Pierce S-Nitrosylation western blot kit, immobilized anti-TMT Resin and TMT elution buffer were purchased from Thermo scientific. Anti-CYR61 was from Abcam. The recombinant plasmid of pcDNA3.1-CYR61 was constructed by our lab.
Cell lines and culture
MDA-MB-231 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA), and were incubated with Leibovitz’s L-15 medium (Catalog No. 30-2008) containing 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin at 37℃ in a free gas exchange with atmospheric air. Human pulmonary microvascular endothelial cells (HPMEC) were purchased from Promocell, and were cultured in ECM with 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin at 37℃ in 5% CO2 atmosphere.
Construction of Si-CYR61 and OE-CYR61 MDA-MB-231 cells
SiRNA oligos and recombinant plasmid pcDNA3.1-CYR61 were constructed by our lab. SiRNA sequence: 5’-AACAUCAGUGCACAUGTAUUG-3’. CYR61 cDNA cloning primer: sense, 5’-taa aag ctt atg agc tcc cgc atc gcc ag-3’ and antisense, 5’-ccc ctc gag tta gtc cct aaa ttt gtg aat gtc-3’. Transient transfection of MDA-MB-231 cells with siRNA oligos (100 pmol) and recombinant plasmid pcDNA3.1-CYR61 (4 µg/well) was carried out using Lipofectamine®3000 Transfection Reagent Protocol (life technologies), according to the manufacturer’s instructions. Nontargeting siRNA and empty pcDNA3.1 vector were served as negative controls, respectively. These cells were harvested 24 h after transfection and used for further analysis.
Biotin switch assay
Biotin-switch assay was performed to verify protein S-nitrosylation [17, 18]. In brief, cell lysates were diluted with HENS buffer (Pierce) and then treated with methyl methanethiol-sulfonate (MMTS) (Pierce) in a final concentration of 20 mM at room temperature. After 30 min, MMTS was removed by passing the samples through a spin column three times. The samples were then incubated with ascorbic acid (Pierce) to release the NO from the thiol group, and were subsequently biotinylated by incubating with biotin-TMT (Pierce). The biotinylated proteins were then precipitated by incubating the samples with 50 µl of neutravidin-agarose (Pierce). The neutravidin-agarose was then pelleted and washed 5 times using HENS buffer. The biotinylated proteins were eluted by TMT-elution buffer (Pierce).
Spectroscopic analysis
MDA-MB-231 s were lysed by RIPA with 1% PMSF, and the cell’s protein concentrations were measured by BCA. Lysates were treated with 1- and 10-fold acidified NO2− for 20 min, respectively, and were then scanned with UV-visible spectroscopy.
S-nitrosylation optimization
The degree of S-nitrosylation (S-nitrosylated protein/total protein) was used to evaluate the trans-nitrosylation reaction between GSNO and proteins. Briefly, MDA-MB-231 s and OE-CYR61 MDA-MB-231 s were cultured with GSNO (10 and 30 µM) for different times and were lysed in HENS buffer. Biotin switch assay was used to label S-nitrosylated protein with -TMT. The total protein amount was measured by BCA assay. The S-nitrosylated proteins were collected by the affinity resin column and quantitatively measured by the BCA assay.
Western blotting assay
Western blotting assay was performed to evaluate protein expression levels as we described previously [19]. Briefly, MDA-MB-231 cells cultured in the present of GSNO (0, 10 and 30 µM) were lysed by RIPA with 1% PMSF. Antibodies used for western blotting analysis included CYR61 antibody and TMT. Immunodetection of electrophoresis-resolved proteins was accomplished using the enhanced chemiluminescence based on the standard protocols. The signal intensity was quantified with a quantitative digital imaging system (Quantity One, Bio-Rad).
S-nitrosylation verification
MDA-MB-231 s were cultured with GSNO, GSH, and GSNO plus DTT (all 30 µM), respectively. Biotin switch assay and western blotting assay were performed to detect protein S-nitrosylation. The S-nitrosylated protein was collected by the affinity resin column. Western blotting assay was performed to detect CYR61 S-nitrosylation.
Determination of S-nitrosylated Cys in CYR61
S-nitrosation of the recombinant CYR61 was determined liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis [20, 21]. Recombinant CYR61 protein (PeproTech) was incubated with GSNO (30 µM) for 30 min at room temperature. Proteins were trypsinized and desalted by using a C18 desalting column, followed by protein lyophilization. The lyophilized powder was dissolved in solution A (100% water, 0.1% formic acid) and injected into a C18 Nano-Trap column (2 cm × 75 µm, 3 µm) and separated by an analytical column (15 cm × 150 µm, 1.9 µm), using a linear gradient elution as listed in Supplementary Table 1. The separated peptides were analyzed by Q Exactive HF-X mass spectrometer (Thermo Fisher), with ion source of Nanospray Flex™ (ESI), spray voltage of 2.3 kV and ion transport capillary temperature of 320 °C. Full scan range from m/z 350 to 1500 with resolution of 60000 (at m/z 200), an automatic gain control (AGC) target value was 3 × 106 and a maximum ion injection time was 20 ms. The top 40 precursors of the highest abundant in the full scan were selected and fragmented by higher energy collisional dissociation (HCD) and analyzed by the MS/MS, where the resolution was 15000 (at m/z 200), the AGC target value was 1 × 105, the maximum ion injection time was 45 ms, the normalized collision energy of 27%, the intensity threshold of 2.2 × 104, and the dynamic exclusion parameter of 20 s. The resulting spectra from each fraction were searched by the search engines: Proteome Discoverer 2.2 (PD 2.2, Thermo). The search parameters were set as follows: mass tolerance for precursor ion was 10 ppm and mass tolerance for product ion was 0.02 Da. Carbamidomethyl was specified in PD 2.2 as fixed modifications. Oxidation of methionine and acetylation of the N-terminus were specified in PD 2.2 as variable modifications. The maximum of 2 missed cleavage sites was allowed. The identified protein contains at least 1 unique peptide with FDR no more than 1.0%.
In vitro cytotoxicity studies
Cell viability was assessed using MTT assay as we described previously [19, 22, 23]. MDA-MB-231 cells were trypsinized and seeded on 96-well plates at 8 × 103 cells/well. After 24-h culture and adhesion, GSNO (0, 10, 50, 100, 200 and 500 µM) was added to the cell culture for another 12-h incubation. Then, MTT (5 mg/ml, 100-µl per well) was added and incubated for 4 hours. The MTT solution was aspirated and replaced with 100 µl/well dimethyl sulfoxide solution (DMSO). After 30-min mixing, the plates were measured at wavelength 570 nm using an infinite M200 Pro microplate reader (Tecan, Switzerland). Each experiment was performed in triplicate.
Heterotypic cell adhesion assay
Quantification of MDA-MB-231 cell adhesion to endothelial cells was carried out as we described previously [3, 23, 24]. Briefly, HPMECs (105 each well) were grown to confluence on 24-well plates. Then TNF-α (final concentration 10 ng/ml) was used to activate HPMECs for 4 hours. GSNO (0, 10 and 30 µM) and Rhodamine 123-labeled MDA-MB-231 cells and their genetically-transfected CYR61 cell lines were co-cultured with the HPMECs monolayers in each well for 2 hours. Non-adhered MDA-MB-231 s were removed by PBS wash. Ten random visual fields for each well were selected and photographed using a fluorescence microscope (Zeiss Axio Observer A1, Germany). Mean inhibition of adhesion for 10 visual fields was calculated by using the equation: % of control adhesion= [the number of adhered cells /the number of adhered cells in the MDA-MB-231 control group] × 100%. Each experiment was performed in triplicate.
Cell-matrix adhesion assay
To conduct a cell-matrix adhesion assay, HPMECs were replaced with gelatins, the later was used to simulate the extracellular matrix. The 24-well plate was coated with 100 µL gelatin per well and incubated for 24 hours. The gelatin-coated 24-well plates were blocked with PBS containing 1% BSA for 30 min before the adhesion assay. BSA was thoroughly removed with PBS for three times. Rhodamine 123-labeled MDA-MB-231 cells and their genetically-transfected CYR61 cell lines were cultured with GSNO (0, 10 and 30 µM) for 2 hours. Non-adhered MDA-MB-231 s were removed by PBS. Ten random visual fields for each well were selected and taken pictures using a fluorescence microscope (Zeiss Axio Observer A1, Germany). Mean inhibition of adhesion for 10 visual fields was calculated by using the equation: % of control adhesion= [the number of adhered cells /the number of adhered cells in the MDA-MB-231 control group] × 100%. Each experiment was performed in triplicate.
Wound healing assay.
Wound healing assay was performed to analyze cell migration in vitro as we described previously[25, 26]. MDA-MB-231 cells and their genetically-transfected CYR61 cell lines were seeded in 12-well plates and incubated to become confluent. Sterile tips were used to scratch the cell layers, which were subsequently washed with PBS for three times, and then cultured with 1 mL of L-15 media containing 2% FBS and different concentrations (0, 10, 30 µM) of GSNO. The cells were photographed by using a fluorescence microscope (Zeiss, Germany) at 0, 24 and 48 h. Each experiment was performed in triplicate.
Cell invasion assay.
Matrigel is the trade name for a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. The transwell invasion assay was conducted as we described previously [25]. The upper chambers of the transwells (24-well, 8 µm pore size) were treated with Matrigel (Becton Dickinson, Waltham, MA, USA) and air-dried. The lower chambers were filled with 750 µL of media containing 20% FBS. MDA-MB-231 cells and their genetically-transfected CYR61 cell lines were seeded at the density of 3 × 104 per well (200 µL) on the upper chambers in L-15 media containing GSNO (0, 10 and 30 µM) and 1% FBS. After 24 hours, the cells that had invaded through the Matrigel membrane were stained with crystal violet, and photographed by using a fluorescence microscope (Zeiss, Germany) (five random fields). Each experiment was performed in triplicate.
Platelet adhesion assay
Platelet adhesion assay was performed as we described previously [27]. Freshly anticoagulated whole blood was centrifuged at 200 × g for 10 minutes, and the supernatant (platelet rich plasma, PRP) was collected followed by centrifugation at 1500 × g for 15 minutes, Platelets were precipitated, and the supernatant was platelet-poor plasma (PPP). Platelets were resuspended and adjusted to the range of 1–3 × 106/µL by diluting with autologous PPP. MDA-MB-231 cells and their genetically-transfected CYR61 cell lines were grown to confluence on 12-well plates, respectively. Then, CFSE-labeled platelets and GSNO (0, 10 and 30 µM) were co-cultured with the MDA-MB-231 s monolayers in each well, followed by addition of ADP (20 µM) to stimulate the platelet activation. After 1-hour incubation at 37 °C, non-adhered platelets and cells were removed by three times of PBS wash. Ten random visual fields for each well were selected and photographed by using the Leica TCS SP8 confocal microscope. The mean inhibition of adhesion for 20 visual fields was calculated by using the equation: % of control adhesion = [the number of adhered cells / the number of adhered cells in the MDA-MB-231 control group] × 100%. Each experiment was performed in triplicate.
Cell morphology assay
MDA-MB-231 cells (2.5 × 104) were cultured with GSNO (30 µM) in a 35-mm cell culture dish (NEST, GBD-35-20). Morphology change of the cells was analyzed by using a time-lapse photography program of the leica TCS SP8 confocal microscope.
Mice
BALB/C nude mice (20 ± 2 g, 4–6 weeks old) were purchased from Shanghai SLAC Laboratory and maintained under clean conditions. Mice were housed in clean, pathogen-free room in an environment with controlled temperature (26 ℃), humidity (55%), and a 12-hour light/dark cycle, and maintained with free access to pellet food and water in microinsulator cages. All animals used in the investigation were handled in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996), and approved by the institutional animal care and use committee of Fuzhou University.
MDA-MB-231 lung metastasis experiment
The nude mice were divided into nine groups (n = 8 per group). The CFSE-labeled MDA-MB-231 cells and their genetically-transfected CYR61 cell lines were pre-treated with GSNO (0, 10 and 30 µM) for 30 min and washed 3 times in PBS. The cells (5 × 106) were resuspended in 200 µl of PBS, and injected into the lateral tail vein of mice. Four hours after the injections, the nude mice were sacrificed and their lungs were excised, and frozen sectioned, and photographed by using the leica TCS SP8 confocal microscope.
In vivo tumor pulmonary metastasis study
The nude mice were divided into treatment groups and control group randomly (n = 8 per group). MAD-MB-231 s were treated with GSNO (0, 10 and 30 µM) for 30 min and washed 3 times in PBS. The cells (5 × 106) were resuspended in 200 µl of PBS, and injected into the lateral tail vein of the mice. Ten weeks after the injection, the nude mice were sacrificed and their lungs were excised. The number of surface lung tumor nodules was counted. The lungs were then paraffin-embedded and stained with hematoxylin and eosin (H&E) for histological examination.
In vivo tumor xenograft study
The nude mice were divided into three groups (8–10 per group). MDA-MB-231 cells treated with or without GSNO were resuspended in 50 µl of PBS, respectively, and injected into mammary fat pad (group1: MDA-MB-231 s and GSNO-treated MDA-MB-231 cells were injected orthotopically into the different mammary fat pad of the same immunodeficient mouse; group 2: MDA-MB-231 s were injected orthotopically into mammary fat pad; group 3: GSNO-treated MDA-MB-231 cells were injected orthotopically into mammary fat pad). Two weeks after the injections, the tumor growth was observed. Six weeks after the injections, the nude mice were sacrificed and their lungs were excised and photographed.
Statistical analysis
Data are presented as the mean ± standard error of the mean (SEM), or mean ± standard deviations (SD). Statistical analysis was performed using the student’s t-test and one-way analysis of variance. A P-value less than 0.05 was considered statistically significant.