The cervical cancer cell lines SiHa and HeLa were cultured in DMEM medium with L-glutamine (Invitrogen, Heidelberg, Germany) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (both from Millipore, Darmstadt, Germany) in a humidified incubator at 37 °C and 5% CO2.
SiHa and HeLa cells were seeded in 96-well plates at a density of 104 cells/well and allowed to recover overnight before initiating the drug treatments. The cells were exposed to different doses of disulfiram (Sigma), CuCl2 (Sigma) and (or) 10 μmol/L cisplatin (DDP, Teva Parenteral, CA) for different times in different experiments as indicated. Cell viability was assessed using the 3-(4,5-Dimethyl-1, 3-thiazol-2-yl)- 2,5-diphenyl-2H-tetrazol-3-ium bromide (MTT; Sigma-Aldrich) assay. Following the manufacturer’s instructions, 20 μl of MTT solution was added into 200 μl of the culture media. The plates were then incubated for 4 h at 37 °C, and the optical density was measured at 490 nm.
Assessment of apoptosis
Apoptotic status was determined by PE Annexin-V/7-AAD Apoptosis Detection Kit I (559,763, BD) using flow cytometry following the manufacturer’s instructions. Briefly, 5 × 105 cells were seeded in T25 flasks for 24 h and treated with drugs for a further 48 h. The cells were rinsed with calcium-containing HBSS and detached using EDTA-free trypsin. The detached cells were washed twice with cold PBS and then resuspended in 100 μl Binding Buffer at a concentration of 1 × 106 cells/ml. All cells were incubated with PE Annexin V (5 μl) and 7-AAD (5 μl) at RT for 15 min in the dark. The cells were diluted in 400 ml of Binding Buffer and then 20,000 events were acquired and analyzed by a FACSCalibur Flow Cytometry (Becton Dickinson, USA). Apoptosis and necrosis were evaluated using FL2 (Annexin V) vs FL3 (7-AAD) plots. The cells stained with Annexin V only were classified as early apoptosis and the Annexin V and 7-AAD double-stained cells were classified as late apoptosis or necrosis.
Cells were lysed in a lysis buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 2 mM EDTA; 1% NP-40; and 0.1% sodium dodecyl sulfate) that contained a protease inhibitor cocktail (Complete Mini; Roche Diagnostics, Branchburg, NJ). The blots were cutted according to molecular size markings prior to hybridisation with antibodies. The membranes were incubated with antibodies against human Bcl2 (1:200 dilution, sc-7382, Santa Cruz Biotechnology), ABCG2 (1:50 dilution, sc-58,222, Santa Cruz Biotechnology), P53 (1:500 dilution, sc-263, Santa Cruz Biotechnology), P27Kip1 (1:500 dilution, sc-528, Santa Cruz Biotechnology), LGR5 (1:200 dilution, Abnova, Taipei, Taiwan) and β-actin (1:1000 dilution, sc-47,778, Santa Cruz Biotechnology) at 4 °C overnight followed by incubation with horseradish peroxidase-conjugated secondary immunoglobulin G (IgG; Thermo Fisher Scientific, New York, NY). The membranes were briefly incubated with an enhanced chemiluminescence reagent (Millipore, Billerica, Mass) and then visualized on X-ray films .
Cell cycle analysis
Analysis of cell cycle progression was performed using DNA staining. Briefly, cervical cancer cells were plated into T-75 flasks at an initial seeding density of 106 cells/flask. After 48 h treatment, trypsinized cells were washed twice with cold PBS, followed by fixation with ice-cold 70% ethanol overnight at 4 °C. After washing twice with PBS, the cells were incubated with 50 μg/ml propidium iodide (Sigma-Aldrich, St. Louis, MO, USA) and 10 μg/ml RNaseA (Sigma) for 30 min at room temperature. The cells were then analyzed using a FACSCalibur (Becton Dickinson, USA) with the FlowJo software (Tree Star Inc., Ashland, USA).
For immunocytochemistry, cells were plated on cover slips, fixed with 4% paraformaldehyde for 30 min at room temperature, and permeabilized with 0.2% Triton X-100 for 20 min at room temperature. The slides were incubated with a rat monoclonal antibody raised against human LGR5 (1:50, Abnova, Taipei, Taiwan) overnight at 4 °C and then with secondary antibodies for 30 min at room temperature followed by diaminobenzidine development. All slides were examined under an Olympus-CX31 microscope and the images were acquired by DP Controller (Olympus, Tokyo, Japan) .
Flow cytometry analysis and FACS isolation of cells
The ALDH enzymatic activity of the cells was measured using the ALDEFLUOR kit (Stem Cell Technologies, Vancouver, BC, Canada), according to the manufacturer’s instructions. The brightly fluorescent ALDH-expressing cells were detected using a FACSCalibur flow cytometer (BD Biosciences). As a negative control, cells were stained under identical conditions after treatment with the specific ALDH inhibitor diethylaminobenzaldehyde (DEAB) . The expression of LGR5 and CD49f in cervical cancer cells was measured using the Alexa Fluor® 647 Rat anti-Human LGR5 (N-Terminal) antibody (562,903, BD) and PE Rat anti-Human CD49f antibody (555,736, BD) according to the manufacturer’s instructions. The data were analyzed using the CellQuest program (BD Biosciences).
Vector construction and transfection
Human full-length LGR5 cDNA was amplified by reverse transcription polymerase chain reaction using mRNA extracted from SW620 cells. The primer sequences were designed as follows:
F5′-CTTCTCGAGCTACTTCGGGCACCATGG AC-3′; and.
The LGR5 DNA fragment was subsequently cloned into the XhoI and SmaI sites of the pCAG-AcGFP vector (Clontech, Mountain View, CA, USA) to generate the pCAG-AcGFP-LGR5 recombinant plasmid. The LGR5-overexpression vectors were transfected into SiHa and HeLa cells using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The transfected cells were treated with G418 (Calbiochem, La Jolla, CA, USA) for 3 weeks, and drug-resistant colonies were collected, expanded, and identified .
In vivo anti-tumor assays
The cervical cancer bearing BALB/c nude mice model was used to investigate the in vivo antitumor efficacy of disulfiram/copper complex. Female BALB/c-nude mice (6–7 weeks old) were obtained from Shanghai SLAC Laboratory Animal Co.,Ltd. (Shanghai, China) and housed in a SPF room that was maintained at a constant temperature (22 °C–25 °C) and humidity (40–50%). The sorted tumor cells (1 × 106) were resuspended in 200 μl of 1:1 PBS/Matrigel (BD Biosciences) solution and injected into the subcutis of the two flanks on the dorsum of each mouse (eight mice in each group). Drug treatment (DSF 30 mg/kg, CuCl2 1.5 mg/kg, DDP 5 mg/kg, 0.9% saline) was initiated after 3 days of cell injection and administered via intraperitoneally injection twice weekly. DSF (6 mg/ml stock) was prepared as follows: 120 mg DSF was dissolved in 1 ml DMSO, made up to 10 ml with PEG300 (Selleck), then added to 10 ml H2O, vortexed and filter sterilized. Animals had access to food and water ad libitum. The tumor size was measured using a vernier caliper every week, and the volume was calculated with the following formula: V = (length×width2) /2.
Statistical analyses were performed using the GraphPad Prism 8.00 software (La Jolla, CA, USA). In the comparisons of 2 groups, Student’s t-test was used to determine statistical significance. To examine differences among > 2 groups, non-parametrical Kruskal–Wallis test or one-way analysis of variance (ANOVA) was used to determine statistical significance. Kaplan-Meier survival analysis was performed, and survival curve comparisons were performed using the log-rank (Mantel-Cox) test. P values of ≤0.05 were regarded as statistically significant.
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