Chemicals and reagents
VALD-3 (purity≥98.0%) was kindly supplied by Professor Song Pengfei (Northwest Normal University, China). Fetal bovine serum (FBS) was purchased from Lonser (Shanghai, China). Primary antibodies against Bax and Bad were obtained from Abcam (MA, USA); and against Bcl-2, cytochrome c, BCL-XL, caspase3, 8, PARP, XIAP, CyclinD1, CyclinB1, CDK-1 and β-actin from Proteintech Group Inc. (Chicago, IL, USA). Primary antibodies to probe the Wnt/β-catenin pathway were obtained from Cell Signaling Technology (Danvers, MA, USA). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), Hoechst 33258 fluorescent dye kit and RIPA cell buffer were obtained from Solarbio Co., Ltd (Beijing, China). The cell cycle and apoptosis kit was purchased from US EVERBRIGHT INC (UE, Suzhou, China). All other reagents and plastic material were obtained from commercial sources.
Human breast cancer MCF-7 and MDA-MB-231 cell lines were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI-1640 medium (HyClone) containing 10% FBS and 1% penicillin streptomycin (HyClone) at 37℃ in a humidified 5% CO2 atmosphere.
Xenograft of human breast cancer cells into nude mice
Six-week-old female BALB/c athymic nude mice (specific pathogen free, SPF) (18 ± 4 g) were purchased from Beijing Weitonglihua Laboratory Animal Co., Ltd. (Beijing, China; animal quality license, SCXK (Jing) 2016-0011). Mice were maintained in sterile conditions at a constant temperature of 22-24°C, 50 -55% humidity, and under a 12 h light/dark cycle. All methods were approved by the Institutional Animal Care and Use Committee of Gansu Provincial Hospital (No. 2017-015). All experiments were conducted in accordance with the guidelines of the Ministry of Health of PR China and the Animal Care Committee of Gansu Provincial Hospital. MCF-7 tumor cells were resuspended at a density of 1.2 × 107 cells/200 µL in saline solution were injected subcutaneously in the front armpit of the nude mice, followed by tumor growth measurements every 3 days. Negative controls were injected with 200 µL of phosphate buffered saline containing no cells. When the tumor size reached 100 mm3, 50 tumor-bearing mice were randomly assigned to the time course and survival experiments, which were conducted simultaneously. The time course experiment included five groups (negative control, control, 5mg/kg/3d cisplatin, 20 mg/kg/d VALD-3 and 5mg/kg/3d cisplatin +20mg/kg/d VALD-3, n=8), whereas the survival experiment included three groups (control, 10 mg/kg/d VALD-3 and 20 mg/kg/d VALD-3,n=6). In the time course experiment, mice were sacrificed by cervical dislocation on day 13 and tumor tissues and blood samples were collected for further analysis. One mouse in the control group died on day 10 due to an oversized tumor. In the survival experiment, mice were treated until they died, the time of death of each mouse was recorded, and the survival analysis curve was plotted. During the course of the experiment, tumor size and body weight were measured every 3 days, and tumor volume was calculated based on the formula length ×width2/2.
The effects of VALD-3 on MCF-7 and MDA-MB-231 cell proliferation were measured with the MTT assay. MTT assay according to our previous test method . Briefly, VALD-3 was dissolved in RPMI-1640 culture media at final concentrations of 0, 2.5, 5, 10, 20, and 40 mg/L. MCF-7 and MDA-MB-231 cells were seeded onto 96-well plates at a density of 3*104 cells /well, incubated at 37℃for 24 h and then treated with various concentrations of VALD-3 (0, 2.5, 5, 10, 20, and 40 mg/L) for 24, 48 and 72 hours. After treatment, 20 µl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT,5mg/ml) was added to each well and cells were incubated for another four hours. Finally, 150µl of DMSO was added to dissolve the formazan crystals. The absorbance at 570 nm was measured with a microplate reader (Molecular Devices, Sunnyvale, USA).
Hoechst 33258 staining
Cells were incubated in 6-well plates, treated for 48h with different concentrations of VALD-3 (0, 2.5, 5, 10, 20, 40 mg/L), washed with PBS and stained with Hoechst 33258 solution in the dark for about 5 min. Stained cells were observed and photographed with an inverted fluorescence microscope (Leica Microsystems, Wetzlar, Germany).
Cell cycle analysis
The effects of VALD-3 on the cell cycle were analyzed in MCF-7 and MDA-MB-231 cells by PI single staining. Briefly, cells were seeded onto 6-well plates. After overnight incubation, cells were treated for 24h with different concentrations of VALD-3 (0, 2.5, 5, 10, 20, 40 mg/L). Next, cells were collected and fixed at 4° C overnight with pre-cooled 70% ethanol. The ethanol solution was discarded and the cells were washed twice with PBS. The proportion of DNA in each sample was determined using a cell cycle kit. Data were acquired on a BD FACS Calibur (BD Bioscience, USA) machine and analyzed using ModFit LT 3.2 software.
Annexin V/PI staining assay
MCF-7 and MDA-MB-231 cells were seeded onto 6-well plates and treated with VALD-3 (0-40 mg/L) for 24, 48, and 72h. The method was carried out according to the instructions of the Annexin V-FITC Apoptosis Assay Kit. Cells were then collected and washed twice with cold PBS. Next, cells were resuspended in 100μL of 1x binding buffer and incubated for 15 min at room temperature in the dark with 5μL of annexin V and 5μL of PI solutions (Annexin V-FITC Apoptosis Assay Kit, BD Biosciences, USA). The percent of apoptotic cells was determined by flow cytometry.
Quantitative real-time RT-PCR (qRT-PCR)
Expression of Bcl-2, Bax, Wnt/β-catenin signaling pathway and its downstream target genes c-Myc, CyclinD1 and DDK-1 was assessed by RT-PCR after VALD-3 (0-40 mg/L) treatment. The primers were designed and manufactured by Takara Bio with the following sequences:Bcl-2 (forward: 5′-GGATTGTGGCCTTCTTTGAG-3′, reverse:5′-TACCCAGCCTCCGTTATCCT-3′), Bax (forward: 5′-CCGATTCATCTACCCTGCTG-3′, reverse: 5′-TGAGCCAATTCCAGAGGCAGT-3′),β-catenin (forward: 5′-CTT ACA CCC ACC ATC CCA CT-3′, reverse: 5′-CCTCCACAAATTGCTGCTGT-3′), c-Myc (forward:5′-GCTGCTTAGACGCTGGATTT-3′, reverse: 5′-GGCATTCGACTCATCTCAGC-3′), cyclinD1 (forward:5′-GCCGAATTCATGGAACACCAGCT-3′, reverse:5′-TGCACCTGTAGACTGAGCTCGC-3′), and β-actin (forward: 5′- GGACTTCGAGCAAGAGATGG -3′, reverse: 5′- AGCACTGTGTTGGCGTACAG -3′). The RT-PCR reaction was performed using the SYBR green detection system (Takara, Japan) and the data were analyzed by the 2-ΔΔCt method.
Western blot analysis
MCF-7 and MDA-MB-231 cells were cultured and treated with VALD-3 (0, 5, 10, 20, 40 mg/L). After 48h, cells were harvested and lysed with lysis buffer (PARP) containing 1% PMSF at 4℃ for 60 min. The protein concentration was quantified with a BCA Protein Assay kit (Solarbio, Beijing, China). Samples were mixed with loading buffer and boiled at 100°C for 10 min. Proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked with TBST containing 5% skim milk for 1.5h at room temperature under constant agitation, followed by overnight incubation with the primary antibodies at 4°C and three washes with TBST for 10 minutes. Membranes were then incubated with the secondary antibodies for another 1.0 h at room temperature. After additional washes, protein bands were visualized by enhanced chemiluminescence (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
Histopathology and immunohistochemistry
Organs and tumors were fixed with 4%paraformaldehyde and embedded in paraffin. The paraffin-embedded specimens were cut into 4 µm thick slides and stained with hematoxylin and eosin (H&E) for pathological analysis. Tumor tissues were immunostained with antibodies specific for Bax and Bad. Images were captured using a light microscope (Nikon, Japan).
Data were analyzed using SPSS version 22.0 software and all results are expressed as the mean ± standard deviation. Statistical differences were assessed by the Student’s t test and one-way ANOVA. A value of p less than 0.05 was considered statistically significant.