The DOC-loaded lipid microbubble (DLLD) was prepared as previously described (28). Briefly, 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine, Distearoylphosphatidylcholine, Dipalmitoyl phosphatidylethanolamine (all from Sigma-Aldrich, Darmstadt, Germany) and DOC (MedChemExpress, Monmouth Junction, NJ, USA) were mixed to a mass ratio of 1:5:2:2. The mixture was dissolved in chloroform and methanol (1:1, v:v) solution. Following rotary vacuum evaporation, glycerol/PBS (1:9, v:v) was added to form DOC/lipid solution (20 mg/ml). Following perfusion with perfluoropropane gas and mechanical vibration, DLLD was obtained. The unembedded DOC was removed by washing in PBS for 15 min at 4°C. The entrapment efficiency of DLLD was determined by Reverse-Phase High Performance Liquid Chromatography (RP-HPLC; Dalian Elite Analytical Instruments Co., Ltd,). The reverse-phase SinoChrom ODS-BP column (200 × 4.6 mm i.d., pore size 5 µm; Dalian Elite Analytical Instruments Co., Ltd.,) was used at a temperature of 30°C. The mobile phase was composed of methanol/acetonitrile/water (50:30:20; v/v) at a flow rate of 1 ml/min. UV absorbance detection was set at 230 nm. The sample quantity was 20 µl. A series of dilutions of DOC (0.1, 0.5, 2.5, 10, 25, 50 and 100 µg/ml) were made to detect the area under the curve, and a linear calibration curve correlating the area under the curve and concentration of DOC was constructed. The concentration of free DOC was calculated according to the linear calibration curve.

The gastric cancer cell line BGC-823 was purchased from the Shanghai Institute of Cell Biology (Shanghai, China) and maintained in DMEM medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% FBS (Thermo Fisher Scientific, Inc.) supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin. In the present study, BGC-823 cells were divided into 4 groups: Control, DOC, DLLD and DLLD + UTMD. In the control group, BGC-823 cells were treated with PBS. In the DOC group, BGC-823 cells were treated with 4 µM DOC. In the DLLD group, DLLD (21.3 mg/l) which was equal to 4 µM DOC was used. In the DLLD plus UTMD group, DLLD-treated cells received ultrasound irradiation (0.5 W/cm², 1 MHz) for 30 sec. Cells were treated with PBS, DOC or DLLD for 48 h at 37°C.

BGC-823 cells were seeded on a 96-well plate at a density of 3×10³ per well and cultured for 24, 48 and 72 h, respectively. A Cell Counting Kit 8 (CCK8; MedChemExpress) was used for cell viability detection. Briefly, cells were incubated with CCK8 (10 µl/well) for 3 h at 37°C. Optical density (OD) was read at 450 nm using a microplate reader. Cell inhibition was calculated according to the formula 1-OD^{expremental group}/OD^{control group}.

A BrdU cell proliferation ELISA kit (Abcam, Cambridge, MA, USA) was used to quantify cells in DNA synthesis. Briefly, cells were incubated with fresh medium containing BrdU solution for 12 h at 37°C. After removing the medium and being washed in PBS for 5 min at room temperature (RT), cells were fixed in 4% paraformaldehyde solution for 10 min at RT and incubated with primary BrdU antibody for 1 h at RT. Cells were subsequently incubated with the secondary antibody for 30 min at RT. Following incubation of cells with TMD and stop solution, the absorbance was determined at 450 nm using a microplate reader (Thermo Fisher Scientific, Inc., USA).

Cells in the different experimental groups were digested using 0.25% trypsin and homogenized by pipetting. For cell cycle analysis, cells were centrifuged at 1,000 × g for 5 min at RT and re-suspended in 70% cold ethanol and stored at 4°C overnight. The ethanol was removed by centrifugation (1,000 × g for 5 min at RT) and cells were washed in PBS for 5 min at RT. Cells were then incubated with propidium iodide (PI; Thermo Fisher Scientific, Inc, USA) for 30 min at 4°C in the dark. Immediately following this incubation, the samples were detected using a flow cytometer (Becton-Dickinson, Heidelberg, Germany). The data were analyzed by FlowJo 7.6 software (Stanford University, California, USA).

For cell apoptosis detection, an Annexin V-FITC Apoptosis Detection kit (Vazyme Biotech, Co., Ltd., Nanjing, China) was used. Following staining with Annexin V and PI for 15 min in the dark, samples were immediately detected using a flow cytometer (Becton, Dickinson and Company). The data were analyzed using FlowJo 7.6.1 software (FlowJo LLC).

For mitochondrial membrane potential (MMP) detection, a JC-1 MMP detection kit (Beyotime Institute of Biotechnology, Haimen, China) was used. Cells treated with carbonyl cyanide 3-chlorophenylhydrazone were set as the positive control. Cells were incubated with JC-1 (5 mg/l) for 1 h at 37°C. The unbound JC-1 was removed by washing in PBS. Samples were immediately detected using a flow cytometer (Becton, Dickinson and Company). The data were analyzed by Flow Jo 7.6 software (Stanford University, California, USA). When the MMP is high, JC-1 aggregates to form a polymeric compound (red fluorescence) in the matrix of mitochondria. On the contrary, JC-1 cannot aggregate and exists as a JC-1 monomer (green fluorescence) (29). Therefore, the MMP was calculated as the ratio of red fluorescence intensity to green fluorescence intensity.

BGC-823 cells were harvested and lysed in RIPA solution (Beyotime Institute of Biotechnology) containing phenylmethanesulfonyl fluoride (PMSF) and phosphatase inhibitor. Following centrifugation (13,000 × g for 15 min at 4°C), the supernatant was collected. The protein content for each sample was determined using the bicinchoninic acid assay method. The protein (25 µg/lane) was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis using a 10% gel. The fractioned proteins in the gel were transferred onto a PVDF membrane. Following immersion in 5% milk/PBST for 1 h at RT, the membrane was incubated with the primary antibodies, including p53 (dilution 1:500; cat. no. ab26), p21 (dilution 1:1,000; cat. no. ab109520), Bcl-2 (dilution 1:1,000; cat. no. ab32124) and Bax (dilution 1:1,000; cat. no. ab32503; all Abcam) at 4°C overnight. The unbound primary antibodies in the membranes were removed by washing in PBS for 15 min at RT. The membranes were then incubated with the corresponding HRP-conjugated goat anti-rabbit (dilution 1:3,000; cat. no. ab6721) and goat anti mouse (dilution 1:3,000; cat. no. ab205719; all from Abcam) secondary antibodies at room temperature for 1 h. Protein bands in the membrane were visualized following staining with enhanced chemiluminescence solution.

Data are expressed as the mean ± SD. One-way ANOVA was used for statistics among groups. When ANOVA was significant, it was followed by a post-hoc Fishers least significant difference test. P<0.05 was considered to indicate a statistically significant difference. The SPSS 19.0 software package was used to perform statistical analyses (IBM Corp.).

To identify an optimal inhibitory dose of DOC on the growth of BGC-823 cells, BGC-823 cells were treated with serial concentrations of DOC (1, 2, 4 and 8 µM) for 48 h. The results demonstrated that DOC significantly inhibited the growth of BGC-823 in a dose-dependent manner, and the optimum inhibition was observed at a dose of 4 µM (Fig. 1A). As a result, 4 µM DOC was subsequently used in the present study.

Cells were treated with vehicle, DOC, DLLD (capsulation efficiency, 76±3.5%) and DLLD plus UTMD for 0, 24, 48 and 72 h respectively. The results demonstrated that treatment with DLLD plus UTMD significantly inhibited the growth of BGC-823 cells compared with DOC or DLLD treatment alone (Fig. 1B). The inhibitory effect of DOC and DLLD alone were similar (Fig. 1B).

Cell cycle analysis revealed that DOC, DLLD and DLLD plus UTMD could significantly decrease the proportion of cells in the S phase and increase it in the G₂/M phase when compared with the control (Fig. 2A). However, treatment with DLLD plus UTMD could further decrease the proportion of cells in the S phase and increase it in the G₂/M phase when compared with treatment with DOC or DLLD alone (Fig. 2A). No significant differences were observed between the DOC and DLLD groups (Fig. 2A). This result was further confirmed by analysis of BrdU incorporation and the expression of cell cycle-regulating proteins. Among the four groups, BrdU incorporative cells were the lowest and the expression of p53 and p21 the highest in the DLLD plus UTMD group (Fig. 2B-E).

The cell apoptosis assay revealed that DOC, DLLD and DLLD plus UTMD could significantly induce apoptosis in BGC-823 cells when compared with the control (Fig. 3A and B). However, treatment with DLLD plus UTMD could further promote cell apoptosis when compared with treatment with DOC or DLLD alone (Fig. 3A and B). The levels of cell apoptosis in the DOC and DLLD treatment groups were similar (Fig. 3A and B).

The results of MMP analysis revealed that treatment with DLLD plus UTMD significantly decreased the MMP level of BGC-823 cells when compared with treatment with DOC or DLLD alone (Fig. 4A). It was also demonstrated that the expression of Bcl-2 was lowest and the expression of Bax highest in the DLLD plus UTMD group (Fig. 4B-D).