H1299 and A549 cell lines were obtained from ATCC (Virginia, USA). H1299 cells were cultured with RPMI 1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA). A549 cells were maintained with DMEM (Gibco, USA) supplemented with 10% FBS. Cells were grown in a humidified 37 °C incubator with 5% CO2. Before treatment, cells were seeded at a density of 1 × 106 cells in a 100-mm dish for 24 hours.
High-intensity focused ultrasound (HIFU)
Cells were processed using a high-intensity ultrasound focusing system (Beijing Yuande FEP-BY02 Ultrasonic Focusing Knife, China). System parameters were set as listed: 1) ultrasonic frequency = 1 MHz ± 10%; 2) input electric power = 0.5~2 kW; 3) focal intensity = 0 W/cm2, 100 W/cm2, 200 W/cm2, 400 W/cm2, 600 W/cm2; 4) fixed irradiation time = 5 s. CDDP was purchased from TargetMol (China).
Cell proliferation was evaluated by an MTT Kit (Sigma-Aldrich, USA) following the manufacturer’s protocol. Cells were divided into four groups: Con, CDDP (10 μM), HIFU (400 W/cm2), and HIFU (400 W/cm2) + CDDP (10 μM). The treated cell suspension was seeded into 96-well plates at a density of 4,000 cells/well. Cells were cultured for indicated times (24, 48, 72, or 96 hours). Then, 20 μL of MTT solution was added to each well and incubated for 4 hours. The culture medium was carefully removed in each well. 150 μL of dimethyl sulfoxide was added in per well, which was shaken at a low speed for 10 min on a shaker. After the crystals were thoroughly dissolved, the absorbance was measured at 595 nm wavelength with the microplate reader (Synergy H4 Hybrid Reader, BioTek, Winooski, USA). Each data point presents the mean ± s.d. of three independent experiments.
Annexin V-APC/7-AAD apoptosis detection kit (KGA1023-1026, KGI) was used to assess the cell apoptosis with FACSCalibur (BD Biosciences, USA) following the manufacturer’s instruction. Cells were cultured in medium containing different CDDP concentrations (0 μM, 5 μM, 10 μM, 20 μM, 40 μM) for 24 hours and then irradiated with an ultrasonic wave of 400 W/cm2 for 5 s. After treatment, cells were collected by centrifugation at 1500 rpm for 5 min and washed with 1× PBS three times, and then incubated with 5 μL of FITC-conjugated Annexin V and 5 μL of PI for 15 min at room temperature in the dark.
A total of 500 cells in different groups were seeded into 6-well plates. Cells with various treatments were cultured for about 14 days in a humidified 37 °C incubator with 5% CO2, the colonies were fixed with 10% formaldehyde for 30 min and then stained with 0.5% crystal violet (Beyotime, China) for 30 min. The well was washed with 1× PBS and air-dried. After drying, the colonies were photographed by the microscope (Olympus, Tokyo, Japan).
Transwell assay was performed for evaluating cell migration and invasion. After different treatments, 1×105 cells in 250 μL of serum-free medium were placed in the upper chamber (8.0 μm pore size; Corning, USA) with a porous membrane for migration assay, while the lower chamber was inserted into a 12-well plate filled with 500 μL medium supplemented with 10% FBS. After 24 hours of incubation at 37 °C, non-migrative cells were removed from the upper surface of the membrane with cotton swabs, and migrative cells on the lower membrane surface were fixed with 4% formaldehyde and stained with 0.1% crystal violet (Beyotime, China). Five random 200× visual fields per well were photographed and calculated under a Nikon Inverted Research Microscope Eclipse Ti microscope. Cell invasion assay was simultaneously conducted as above, except for the chambers with Matrigel solution (BD, USA).
Determination of cellular Pt accumulation
1 × 106 cells were seeded in 100-mm tissue culture dishes (Corning, USA). After 24 hours, cells were exposed to freshly dissolved CDDP (10 μM). After 1 hour of CDDP drug exposure, the medium was removed and the cells were washed with ice-cold 1× PBS, scraped and collected in double-distilled water. Samples were freeze-dried overnight and Pt was solubilized in HNO3 following the standard procedures. Total Pt content was measured by flameless absorption spectroscopy (FAAS) (Model 3300, PerkinElmer, Norwalk, CT). Under these conditions, the detection limit was 10 mg/L. Pt levels were expressed as nmol Pt/106 cells, with cell number assessed by counting parallel cultures.
Western blot analysis
Cells in each group were collected and lysed in RIPA buffer (Beyotime, China). The protein concentration was determined by a BCA Protein Assay kit (Sangon Biotech, China). 30 μg protein in each group was separated on 10% SDS-PAGE. The proteins were transferred to a polyvinylidene fluoride membrane and blocked with 5% non-fat milk for 1 hour at 4 °C. The membranes were then incubated with primary antibodies, anti-GAPDH (Abcam, USA) (ab9485, 1:2500 dilution) and anti-cleaved-PARP (Abcam, USA) (ab32064, 1:1000 dilution) at 4 °C overnight. Anti-rabbit IgG was used as the secondary antibody for 1 hour at room temperature. The results were finally detected by ECL Chemiluminescence Kit (Thermo Fisher Scientific, USA). The bands were obtained by GeneGnome 5 (Synoptics Ltd., UK).
Tumor xenograft experiments
Animal experiments were formally approved by the Department of Ultrasound, the First Affiliated Hospital of Xinxiang Medical College Ethics Committee and performed in accordance with the 3R guidelines followed for the welfare of laboratory animals. Nude BALB/c male mice (5 weeks old) were used in this study. Sufficient water and food were given during the experiment. As previously reported, fresh lung cancer tissue from an NSCLC patient, approximately 2×2×2 mm3 in size, was inoculated into the abdominal wall of one side of nude BALB/c mice. The mice were divided into four different groups: Con (n = 6), CDDP (2 mg Pt/kg), HIFU (1000 W/cm2), HIFU (1000 W/cm2) + CDDP (2 mg Pt/kg). Saline or CDDP was injected on days 0, 6, 12, 18, and 20, respectively. HIFU-treated mice were processed using a high-intensity ultrasound focusing system (Beijing Yuande FEP-BY02 Ultrasonic Focusing Knife, China) every six days. System parameters were set as listed: 1) ultrasonic frequency = 1 MHz ± 10%; 2) input electric power = 0.5~2 kW; 3) focal intensity = 1000 W/cm2; 4) fixed irradiation time = 5 s. Tumor volumes were measured weekly using a vernier caliper and recorded every four days when the tumor volume reached about 80 mm3. Tumor volume was calculated using the formula: Tumor volume (mm3) = (height) × (width)2/2. Besides, the survival rates of each group were recorded and analyzed every four days. After 20 days, mice were sacrificed for further analysis, and tumor weights were recorded.
Statistical analysis was employed using SPSS software. The significance of difference between the two groups was calculated with Student’s t-test; One-way ANOVA by Tukey’s post hoc test was used to determine the significance among over two groups. P values less than 0.05 were considered significant (*, P < 0.05; **, P < 0.01). The error bars show the means ± s.d. of three independent biological experiments.