Cell lines and culture
A panel of six bladder cell lines (BIU-87, EJ-1, J82, SCaBER, T24 and 5637) and one non-small cell lung cancer cell line (A549) were purchased from the American Type Culture Collection (Manassas, VA, USA). All cell lines were maintained on RPMI-1640 medium (Gibco®, Life Technologies, Carlsbad, California, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco®, USA) in a humidified atmosphere of 5% CO2 at 37°C.
Reagents
BCT-100 was kindly provided by Bio-cancer Treatment International Limited, Hong Kong. N-Acetyl-L-cystein (NAC, Cat No, A7250) and chloroquine (CQ, Cat NO, C6628) were purchased from Sigma. MK-2206 (Cat NO, SF2712-5mg) and Rapamycin (Cat No, S1842-25mg) were bought from Beyotime Biotechnology.
Western blot analysis
Treated cells were harvested, washed and resuspended in NP-40 lysis buffer (Beyotime Biotechnology, Jiangsu, China) with addition of protease inhibitor (1 mM phenylmethylsulphonyl fluoride) for 1hr. A tissue protein extraction reagent kit purchased from Thermo was used to extract protein from xenografts. Supernatants were collected after centrifugation (13000 rpm, 4°C, 30 min). For mitochondrial and cytosolic protein extraction, cell mitochondria isolation kit was purchased from Beyotime Biotechnology. Protein concentration was measured by Bradford Protein Assay Kit (Bio-Rad, Berkeley, CA, USA). 30-60 µg protein was loaded into 8-15% sodium dodecyl sulfate polyacrylamide gel electrophores (SDS–PAGE) and electroblotted onto polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA). The membranes were blocked for 1hr at room temperature in PBS containing 5% non-fat dry milk plus 0.1% Tween-20 (PBST) and incubated overnight at 4°C with monoclonal or polyclonal primary antibodies [ASS1, OTC, cleaved PARP (Santa Cruz Biotechnology, CA, USA), PEG (RevMAb, San Francisco, USA), b-Actin (Sigma-Aldrich), cytochrome C, Smac, COX IV, p-AKT, AKT, p-mTOR, mTOR and Survivin (Cell Signaling Technology, MA, USA)]. Horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology, MA, USA) were applied and membranes incubated for 1hr at room temperature. Detection was conducted using an enhanced chemiluminescence (ECL) kit (GE Healthcare). Quantification was performed using GelQuantNET software (Biochem Lab Solutions, CA, USA).
Cell viability assay
SCLC cells were seeded in a 96-well plate, approximately 104 per well. After drug exposure, 10 µL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (0.5 mg/mL) (Sigma–Aldrich) was added to each well for 3hr, followed by triple lysis solution (10% Sodium dodecyl sulfate, 5% isobutanol, 0.012 mol/L HCl in water), 100 µL per well for 2hr. Optical density was measured at 570 nm using a microplate reader Fluo Star Optima (BmgLabtec GmbH, Ortenberg, Germany).
Arginine concentration detection
The arginine level in cells, serum and tumor was measured using an L-arginine ELISA kit purchased from Abcam (Cat NO, ab241028). The procedure was performed according to the manufacturer’s instructions. In brief, derivatized samples and standards were incubated with L-arginine antibody overnight at 4°C, followed by incubation with peroxidase conjugate for 1 hr at room temperature, tetramethybenzidine substrate for 10 min in the dark. Wash buffer was needed between every step and stop solution added to halt the reaction. Absorbance (450 nm), using a reference (620 nm), was measured using a microplate reader Fluo Star Optima.
Short hairpin RNA (shRNA) transfection
Silencing of ASS1 (shRNA) was using a lentiviral particles kit bought from Santa Cruz. In brief, pretreatment was with polybrene prior to addition of lentiviral particles and incubation for 24 hr. Mixture was then removed to complete medium for a further 48hr incubation. Puromycin dihydrochloride was used to select the stable ASS1-silenced cell line. Corresponding protein was detected by Western blot and cell viability after shRNA was confirmed by MTT assay.
Autophagic flux analysis
Autophagic flux in T24 cell was detected by using the mRFP-GFP-LC3 adenovirus (Hanbio, China). After plating the cells in a 6-well plate at a density of 1×105cells/dish and incubating with mRFP-GFP-LC3 adenovirus for 24 hr, the cells were treated with or without BCT-100 (20 mU/mL) for 24 hr. Autophagic flux was observed under an inverted fluorescent microscope (Zeiss, Germany). The yellow puncta indicated autophagosomes, and the red puncta indicated autolysosomes.
Flow cytometry of Annexin V/PI staining
Apoptosis was determined by flow cytometry using a FITC-conjugated annexin V/PI kit (Beyotime Biotechnology, Jiangsu, China). Briefly, cells were harvested, washed, and resuspended in binding buffer provided in the kit. After 15 min incubation of annexin V/PI at room temperature, samples were measured using a BD FACSAria II analyzer with FL2/FL4 channels (BD, New Jersey, USA).
Reactive oxygen species (ROS) measurement
2',7'-dichlorodihydro-fluorescein diacetate (H2DCFDA) (Beyotime Biotechnology, Jiangsu, China) were employed to test hydrogen peroxide (H2O2). Briefly, treated cells were harvested, washed and incubated with H2DCFDA (1 µM) in medium without FBS for 30 min at 37˚C, washing two times following flow cytometry analysis. GSH content was measured by 5-chloro-methylfluorescein diacetate (CMFDA) (Invitrogen). Treated cells were collected, washed and incubated with CMFDA (5 µM) for 30 min at 37˚C in FBS-free medium, then changed to complete medium for a further 30 min incubation prior to flow cytometry analysis.
Tumor suppression effect of BCT-100 using xenograft models
Xenograft models were established by subcutaneous injection of 107 cells into nude mice (4-5 weeks, 10-14g, female, BALB/cAnN-nu, Laboratory Animal Centre of Shenzhen University, Guangdong, China). When the tumor reached a mean group size of 40–60 mm3, mice were randomized to one of three groups. PBS and BCT-100 (20 and 60 mg/kg) were administered intraperitoneally twice a week. Tumor size was calculated according to the following formula (Size = Length × Width × Depth/2) (21). The relative tumor volume (RTV) was calculated by the formula: RTV = Vn/V0, where Vn was tumor size on day n and V0 was tumor size on the first day of treatment. Mice were sacrificed when tumor volume reached 600 mm3, which was regarded as a humane endpoint. The study protocol was approved by the laboratory animal ethical committee of Guangdong Medical University.
Terminal deoxynucleotidyl transferase-dUTP Nick End Labeling (TUNEL) assay
TUNEL assay was conducted using a Click-iT® Plus TUNEL Assay kit (Beyotime Biotechnology, Jiangsu, China) according to the standard protocol provided by the manufacturer. Cell crawling, fixation and permeabilization were required before TUNEL assay. Cells were immersed in terminal deoxynucleotidyl transferase (TdT) reaction buffer, then changed to new TdT reaction buffer that contained EdUTP, TdT and TdT enzyme. Samples were incubated with TUNEL reaction cocktails followed by 4’,6-diamidino-2-phenylindole (DAPI, Life Technologies) staining. Images were captured using a Zeiss fluorescence microscope.
Immunofluorescence staining
Fixed and permeabilized cell or tumor sections were blocked with 3% bovine serum albumin (BSA) for 1 hr at room temperature, followed by incubation with ki67 antibody overnight at 4˚C. After washing with PBST for 30 min, Alexa Fluor anti-rabbit (Life Technologies) antibody was applied followed by incubation for 1 hr protected from light. The slides were mounted with Prolong® Gold anti-fade reagent with DAPI (Life Technologies). Pictures were obtained using a Zeiss fluorescence microscope.
Statistical analysis
All data were obtained from at least three independent experiments and are shown as mean ± standard deviation (SD). Statistical analysis of data was performed using Student’s two-tailed t-test by Prism5 (GraphPad Software, La Jolla, Southern California, USA).