The RPMI 1640 and trypsin-EDTA solution were purchased from Gibco; Thermo Fisher Scientific, Inc., (Waltham, MA, USA). The fetal bovine serum (FBS) was purchased from Bovogen Biologicals Pty Ltd., (Keilor East, Victoria, Australia). The Cell Counting Kit-8 (CCK-8) was purchased from Beijing Zoman Biotechnology Co., Ltd., (Beijing, China) and the bicinchoninic acid kit was purchased from Multi Sciences Co., Ltd., (Shanghai, China). The polyvinylidene fluoride (PVDF) membranes were purchased from Roche Applied Science (Penzberg, Germany). The antibody against β-actin (polyclonal rabbit anti-mouse) was purchased from Hangzhou HuaAn Biotechnology Co., Ltd., (Hangzhou, China). The antibodies against B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), caspase-3 and caspase-9 (all polyclonal rabbit anti-mouse) were purchased from Arigo Bio (Taiwan, Xinzhu). The secondary fluorescence anti-body (polyclonal goat anti-rabbit HRP) was purchased from KPL, Inc., (Gaithersburg, MD, USA). The sansalvamide analogue LY-15 was developed in Hebei Province Key Laboratory of Molecular Chemistry for Drug (Shijiazhuang, China).

B16 cancer cells were selected for the present study. The B16 cell line was obtained from the Research Center of the Fourth Hospital of Hebei Medical University (Shijiazhuang, China). The cells were grown in RPMI 1640 medium with 10% heat-inactivated FBS and 100 µg/ml penicillin and streptomycin. The cell line was grown in a humidified atmosphere of 95% O₂ and 5% CO₂ at 37°C and the cells were periodically seeded into 25 cm² flasks. The media was changed every second or third day. For the experiments, the cells were grown to 80–90% confluence, digested with trypsin-EDTA, and plated in 25 cm² flasks, and the media was changed every second or third day on 6- or 96-well plates.

LY-15 was dissolved in DMSO and diluted with serum-free medium to prepare solutions of 1,000, 100, 10 and 1 µM. Single-cell suspensions of B16 cells were prepared and adjusted to the indicated concentrations. The cells were inoculated in 96-well plates (100 µl/well) with 5,000 cells/well. After overnight inoculation for cell adherence, the old medium was discarded and replaced with fresh medium with different concentrations of 100, 50, 25, 15, 10, 5 and 1 µM. Each group was placed into six wells, and a 1% DMSO group was simultaneously prepared as the control. The CCK-8 method was used to calculate the percentage growth of the B16 cells treated with various concentrations of LY-15 for 24 h.

Upon reaching 80% confluence, the cells were digested with trypsin-EDTA and serum-free medium was used to make a single-cell suspension. The cells were seeded over night in 96-well plates at a concentration of 3,000 cells/well. The wells were then replaced with fresh complete medium and treated with 10 µM LY-15. The percentage growth of the B16 cells treated for 24, 48, 72, 96 and 120 h was calculated.

Five uniform lines were drawn behind the 6-well plates using a marker pen. The single-cell suspension was seeded in the 6-well plates at a concentration of 200,000 cells/well. After 6 h, 20 µl pipette tips were used to draw through the marker lines. The wells were washed with PBS thrice and fresh media (2 ml/well) with different concentrations at 1, 2, 5 and 15 µM were added; moreover, 1% DMSO was added to the last well that was simultaneously prepared as the control. Images were captured at 0 and 24 h in the same position. We examined the effect of LY-15 on B16 cell migration using the cell scratch test.

At 80–90% confluence, the cells were treated with 2, 5, 10, 15 and 25 µM LY-15 for 24 h, and a control group was prepared. The treated and untreated cells were harvested, washed twice with PBS, and mixed with 1xbuffer 100 µl. After blending the cells, 10 µl FITC and 5 µl propidium iodide (PI) were added. The cells were kept in a dark place for 30 min.

The cells at 70–80% confluence were treated with 5, 15 and 25 µM of LY-15 for 24 h, and a control group was prepared. Proteins were separated using SDS-polyacrylamide gel electrophoresis. Equal amounts of protein (50 µg/sample) from B16 cells treated with LY-15 were loaded to 10% SDS-PAGE in an electrophoresis buffer in a Bio-Rad slab gel apparatus. The proteins were then transferred to a PVDF membrane under the conditions of 80 V for 20 min, 100 V for 90 min and 200 mA for 120 min. Next, the membranes were incubated in antibody dilution solution (rabbit anti-mouse bax, bcl-2, caspase-3 and caspase-9; 1:1,000 and β-actin; 1:3,000) overnight at 4°C. The blots were then incubated with the secondary antibody (1:3,000) for 1 h at 37°C. Results were obtained using the Aplegen Omega Lum C Gel Imaging System (Gel Company, Inc., San Francisco, CA). Concetration of protein was calculated by Image J (NIH, USA).

Statistical analysis was performed using SAS software (SAS Institute, Inc., Cary, NC, USA) and R programming language. Values were expressed as the mean ± standard error and were analyzed using one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were conducted using Graphpad Prism v5 (GraphPad Software, Inc., La Jolla, CA, USA).

The apoptosis and death of B16 cells were induced by the LY-15 treatment in various concentrations of (1, 5, 10, 15, 25, 50 and 100 µM) for 24 h. Moreover, the proliferation rate of the B16 cells showed a decreasing trend. Compared with the control group, no significant difference was identified for the proliferation rate of the 1% DMSO group (P>0.05), whereas those for the treatment of the B16 cells with 100, 50, 25, 15 and 10 µM LY-15 significantly decreased (P<0.001 for all treatment groups, Fig. 2). The morphological changes in B16 cells were observed via optical microscopy (Fig. 3). The B16 cells treated with 15 µM LY-15 for 24 h exhibited morphological changes, including decreased cell density, separation of the adjacent cells, and cell shrinkage.

The proliferation rate of the B16 cells with the treatment of the concentration of 100, 50, 25, 15, 10, 5 and 1 µM LY-15 was investigated. The concentration 10 µM LY-15 was significantly reduced in a time-dependent manner compared with that of the control group (P<0.001 for all time-dependent groups, Fig. 4). The results showed that LY-15 time-dependently inhibits the growth of B16 cells.

Compared with the control group, no obvious difference was identified in the cell migration of the 1% DMSO group. The cell migration of B16 was weakened by the LY-15 treatments with concentrations of 1, 2, 5 and 15 µM for 24 h. The ability of cell migration gradually decreased as the concentration increased. The cell migration changes were observed under optical microscopy (Fig. 5). The cells lost their ability to migrate at LY-15 concentration of 15 µM.

The ability of LY-15 to induce apoptosis was revealed by analyzing the cell samples using flow cytometry. The cells in the early stage of apoptosis were detected by Annexin V, whereas those in the late apoptosis were assessed by PI staining. The 1% DMSO group did not show significant difference in apoptosis compared with the control group. The early and late apoptosis stages of the B16 cells gradually increased with the treatment of LY-15 (5, 15 and 25 µM) for 24 h (Fig. 6). The percentage of apoptotic cells in the control group was 1.46; however, the proportion of apoptotic cells reached 8.57 in the group treated with 15 µM LY-15 for 24 h.

At the present stage of the study, the results of the western blot analysis showed an increasing trend in the expression of Bax, whereas that of Bcl-2 showed the opposite. Caspase-3 and caspase-9 expressions were analyzed using western blot analysis and further confirmed that LY-15 induces apoptosis. The expression levels of caspase-3 and caspase-9 in the B16 cells increased with the treatments of 5, 15 and 25 µM LY-15 (Fig. 7). All results revealed that LY-15 induces the B16 cells apoptosis through a mitochondrial pathway.