Bergamottin was dissolved in dimethyl sulfoxide (DMSO) at a stock solution of 100 mM and stored at −20°C. Dulbecco's modified Eagle's medium (DMEM), RPMI-1640 medium, propidium iodide (PI), Triton X-100, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), penicillin/streptomycin solution and Hoechst 33342 were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Fetal bovine serum (FBS) was obtained from Gibco-BRL (Grand Island, NY, USA). Annexin V-FITC/PI apoptosis detection kit was purchased from Beyotime Institute of Biotechnology (Shanghai, China).

The fruit of C. bergamia was collected from July to August 2014 from Zhengzhou City, China and identified by Professor Ying Lin. A voucher specimen (18-097-018-14) was deposited in the Herbarium of Southeast University, Nanjing, China. A hot extraction procedure was used to prepare the extract. The vacuum dried, finely powdered peel of the fruit (4 kg) was extracted for 48 h with chloroform in a soxhlet apparatus to yield the extract, which was concentrated under reduced pressure. The chloroform extract (50 g) was loaded onto a silica gel (60–120 mesh, 200 g) column and eluted with an increasing gradient of hexane and dichloromethane. Fractions of 100 ml volume each were collected and pooled according to TLC analysis. Five major fractions were collected (90:10, 80:20, 70:30, 60:40, 50:50 and 40:60). The fraction (hexane, dichloromethane; 70:30) yielded bergamottin as a colorless crystalline solid which was identified by ¹H and ¹³CNMR in comparison with previous studies (12).

Human lung adenocarcinoma cancer A549 cells were obtained from the Shanghai Institute of Cell Resource Center of Life Science (Shanghai, China). Cells were grown in DMEM supplemented with 10% FBS and 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were cultured in a CO₂ incubator (New Brunswick Galaxy 170R; Eppendroff) with an internal atmosphere of 95% air and 5% CO₂ gas and the cell lines were maintained at 37°C. The media were stored at low temperature (2–8°C) and the medium for cryopreservation contained 20% phosphate-buffered saline (PBS) and 10% DMSO in growth medium.

Inhibition of cell proliferation by bergamottin was measured by the MTT assay. Briefly, the cells were plated in 96-well culture plates (1×10⁵ cells/well). After 24 h of incubation, the cells were treated with bergamottin (0, 5, 10, 25, 50, 75 and 100 µM) for 24 and 48 h, MTT solution (10 mg/ml) was then added to each well. After a 4-h incubation, the formazan precipitate was dissolved in 100 µl DMSO, and then the absorbance was measured in an automated micro-plated reader (Bio-Tek, Winooski, VT, USA) at 570 nm. The cell viability ratio was calculated by the following formula: Inhibitory ratio (%) = (OD_control − OD_treated)/OD_control × 100%. Cytotoxicity was expressed as the concentration of bergamottin needed to inhibit cell growth by 50% (IC₅₀ value).

Cells were suspended in 1 ml of DMEM containing 0.5% agarose (Amresco, Solon, OH, USA) and 10% FBS, and plated on a bottom layer containing 0.8% agarose and 10% FBS in 6-well plate in triplicate. The cells were treated with 0, 5, 10, 25 and 50 µM of bergamottin for 48 h. After 1 week, the plates were stained with 0.3% gentian violet, and the colonies were counted under a light microscope (13). In each plate ~500 cells were chosen and observed under the microscope.

The invasion assay was carried out in a 24-well plate. Matrigel (BD) coating was carried out on a polyvinylpyrrolidone-free polycarbonate filter (6-mm pore size). The lower chamber was filled with medium containing 10% FBS. A549 cells (1×10⁵ cells/well) were pre-incubated with different bergamottin concentrations (0, 5, 10, 25 and 50 µM) for 20 min at room temperature and the cell medium containing bergamottin was seeded onto the upper chamber wells. Following incubation for 48 h, the filter was fixed and stained with 3% ethanol containing 0.3% crystal violet for 20 min. The stained cells were counted under a light microscope.

The wound healing assay was performed using a standard method (14). Cells (1×10⁶ cells/ml) were seeded in a 6-well plate and incubated at 37°C until a 95–100% full confluent monolayer was obtained. Subsequent to 12 h of starvation, a 100-ml pipette tip was used to create a straight cell-free wound. Each well was washed twice with PBS to remove any debris and then exposed to various concentrations of bergamottin (0, 10, 25 and 50 µM) in a medium. After 48 h of incubation, the cells were fixed and stained with 3% ethanol containing 0.5% crystal violet powder for 20 min, and randomly chosen fields were photographed under a light microscope. The number of cells that migrated into the scratched area were counted.

A549 cells were seeded into 6-well plates at a density of 1×10⁶ cells/well in 1 ml medium. The cells were treated with the different concentrations (0, 10, 25 and 50 µM) of bergamottin for 48 h. The morphological changes were observed and the images were captured under an inverted light microscope (Olympus; Olympus Optical Co., Ltd., Tokyo, Japan) after 48 h. The same spot of cells was marked and captured. The images were captured at a magnification of ×200.

A549 cells were seeded into 12-well plates at a density of 1×10⁶ cells/well in 1 ml culture medium. After treatment with the different concentrations (0, 10, 25 and 50 µM) of bergamottin for 48 h, cell apoptosis was determined by the Hoechst staining kit according to the manufacturer's instructions. After the treatment, cells were fixed with 4% polyoxymethylene and then incubated in Hoechst solution for 10–15 min in the dark. The staining images were recorded using a UV fluorescence microscope (Olympus) using a UV filter at a magnification of ×200 to detect morphological evidence of apoptosis.

Apoptotic cells were quantified using an Annexin V-fluorescein isothiocyanate (FITC)/PI kit (BD Biosciences, San Jose, CA, USA) and detected using flow cytometry using a FACSCalibur flow cytometer (Becton-Dickinson and Company, Franklin Lakes, NJ, USA) and analyzed using ModFit and CellQuest™ software. A549 cells were plated at a density of 1×10⁶ cells/well into 12-well plates and incubated overnight. The cells were then treated with bergamottin at varying doses (0, 10, 25 and 50 µM) for 48 h. Cells grown in media containing an equivalent amount of 0.11% DMSO without any drug served as the control. Cells were then collected and resuspended in binding buffer. Cells were incubated with Annexin V-FITC and PI for 30 min in the dark, previous to flow cytometric analysis. Annexin V-positive cells were considered to be in the early stage of apoptosis, whereas Annexin V and PI-positive cells were considered to be in the late stage of apoptosis.

Briefly, A549 cells (1×10⁶ cells/ml) were seeded into each well of 6-well plates and incubated for 24 h for cell attachment and recovery. The cells were treated with different concentrations (0, 10, 25 and 50 µM) of bergamottin for 48 h. After incubation for 24 h, the cells were harvested and fixed with ice-cold 70% ethanol (2 ml) at −20°C for 1 h. Prior to analysis, the cells were washed with cold PBS and re-suspended in 450 µl of PBS, 25 µl PI and 25 µl RNase A. The DNA contents were recorded by a FACSCalibur flow cytometer equipped with Cell Quest software.

The effect of bergamottin on mitochondrial membrane potential in human non-small cell lung cancer cells (A549) was detected using the Rhodamine 123 (5 mM) fluorescent probe. A549 cells (2×10⁵ cells/dish) were treated with different concentrations of bergamottin (0, 10, 25 and 50 µM) for 48 h. Rhodamine 123 (2 mM) was added 2 h before the termination of the experiment. Mitochondrial membrane potential was measured by flow cytometry, a BD FACSCalibur flow cytometer and Cell Quest software 3.0 (Becton-Dickinson).

The effects of bergamottin on tumor progression were also observed using a nude mouse model. Female BALB/c nude mice (six weeks old) were purchased from the Shanghai Laboratory Animal Center (SLAC; Shanghai, China). All mice (a total of 20 were obtained) were maintained with water and food ad libitum in a pathogen-free environment with a 12 h light and 12 h dark cycle in an animal care facility and according to animal welfare regulations and protocols approved by the Affiliated Tumor Hospital of Zhengzhou University/Tumor Hospital of Henan Province, Zhengzhou, China. Human non-small cell lung carcinoma A549 cells (2×10⁶ cells/mouse) were injected into the right axilla of the nude mice (5 mice/group) to create tumors in the mice. Subsequent to tumor development, the mice were divided into 4 groups and treated with bergamottin injected intraperitoneally. The control group in the study was treated with an equal amount of PBS while the other three groups were treated with 25, 50 and 100 mg/kg of bergamottin. Afterwards, the mice were sacrificed after 18 days, and the tumor weight and volume of each mouse were evaluated. Tumor length and width were measured using a Vernier caliper (Fisher, Pittsburgh, PA, USA) and the tumor volume (TV) was calculated using the formula: TV = length × width × 0.5 width.

All data were derived from at least three independent experiments. The results are expressed as the mean ± SD. Differences between groups were analyzed using the Student's t-test. P<0.05 was considered to indicate a statistically significant result.

The chemical structure of bergamottin is shown in Fig. 1. Initially we demonstrated the antiproliferative activity of bergamottin on A549 cells using MTT assay. The results revealed that bergamottin had potent antiproliferative effects on the A549 cells. Bergamottin showed both concentration-dependent as well as time-dependent growth inhibitory effects against these cells (Fig. 2). Bergamottin also inhibited the clonogenic activity of the A549 cancer cells by reducing the number of cancer colony forming cells. A reduction in clonogenicity also followed the concentration dependence on bergamottin (Figs. 3 and 4). The results indicate that bergamottin has the tendency to inhibit both cell proliferation (anchorage-dependent) and colony formation (anchorage-independent) growth of NSCLC cells in vitro. Paclitaxel at 5 µM was used as a positive control.

Lung cancer is very lethal since it is highly invasive particularly in later stages. In this part of the study, we demonstrated whether bergamottin could inhibit the invasive behavior of the NSCLC A549 cells. The invasive assay was designed using A549 cells using Matrigel-coated 24-well microchemotaxis chambers in the presence of bergamottin at various concentrations. As shown in Figs. 5 and 6, bergamottin at various concentrations (0, 5, 10, 25 and 50 µM) considerably inhibited the invasion of the A549 cells in a dose-dependent manner. Paclitaxel was used as a positive control and it was observed that the inhibition of cell invasion induced by bergamottin at higher doses was comparable to paclitaxel.

In this experiment, we evaluated the effect of bergamottin on the cell migration in NSCLC A549 cells. Confluent cells were scratched and then treated with bergamottin in a complete medium for 48 h. The number of cells that migrated into the scratched area was photographed (magnification, ×40) and calculated as a percentage (%) of migration. As shown in Figs. 7 and 8, bergamottin markedly reduced A549 cell migration in a concentration-dependent manner. Fig. 8A represents untreated (0 µM) control cells while Fig. 8B–D represent the effect of a 10, 25 and 50 µM dose of bergamottin, respectively. Paclitaxel at a dose of 5 µM was used as a positive control.

In the present study, the morphological alterations in the human NSCLC A549 cells untreated and treated with bergamottin were observed under an inverted light microscope. The most conspicuous changes characteristic of apoptosis were observed in the treated cells that included the detachment of the cells from the substratum and cell shrinkage. As revealed by inverted light microscopy, the untreated control cells were evenly distributed on the substratum. A decrease in the cell population was noted with the increase in the bergamottin concentration. As shown in Fig. 9A–E, the untreated A549 cells appeared as densely packed and disorganized multilayers, whereas after incubation with various concentrations of bergamottin for 48 h several of the cells became rounded and shrunken, and detached from each other or floated in the medium. Paclitaxel was used as a positive control and its effect is shown in Fig. 9E.

The apoptosis-inducing effect of bergamottin was further assessed by Hoechst 33258 staining using fluorescence microscopy. Following the treatment with different doses of bergamottin (0, 10, 25 and 50 µM) for 48 h, the cells were analyzed by fluorescence microscopy. Bergamottin-treated cells stained with Hoechst 33258 revealed chromatin condensation, fragmented nuclei and nuclear shrinkage which increased with the increasing dose of bergamottin (Fig. 10A–E). The number of apoptotic cells increased to 43.2 and 66.4%, respectively, at 25 and 50 µM bergamottin dose for 48 h. At a lower dose of 10 µM, no significant increase in apoptotic cells was noted. Paclitaxel was used as a positive control and its effect is shown in Fig. 10E.

Flow cytometry was employed to demonstrate the effect of bergamottin on cell cycle phase distribution in the lung cancer A549 cells. The results showed that the population of cells in the G2/M phase of the cell cycle increased from 1.89% in the control (untreated cells) (Fig. 11Aa) to 15.21, 28.31 and 52.12% in the A549 lung cancer cells treated with 10, 25 and 50 µM of bergamottin, respectively (Fig. 11Ab–d). Cell cycle arrest and apoptosis may result from extensive DNA damage caused by bergamottin in the A549 cancer cells. A higher number of A549 cells accumulated in the G2/M phase after treatment with increasing concentrations of bergamottin. Fig. 11B shows the graphical representation of the G2/M phase cell population at different bergamottin concentrations.

Rhodamine 123 is one of the most commonly used dyes for measuring mitochondrial membrane potential (MMP) loss in cells. In the present study, we evaluated the effect of bergamottin on MMP loss in NSCLC cancer cells using flow cytometry. The results of the effect of bergamottin on mitochondrial membrane potential are shown in Fig. 12A–E, which revealed that bergamottin induced a dose-dependent loss of MMP in the A549 cells. The number of cells with intact mitochondrial membrane potential decreased by 32.8 and 52.3%, respectively, at a 25 and 50 µM dose, respectively. Fig. 12A represents untreated cells, while Fig. 12B–E represent cells treated with 10, 25 and 50 µM and paclitaxel (5 µM), respectively.

From the in vitro experiments, it was confirmed that bergamottin exerts potent cytotoxic effects inhibiting cell proliferation and inducing apoptosis. However, there are many instances where a drug shows bioactivity in in vitro experiments but fails when used for in vivo experiments. Therefore, we also determined the anticancer efficacy of this compound under in vivo conditions using female BALB/c nude mice (a total of 20 mice were used). Tumors were induced in the mice by injecting non-small cell lung cancer A549 cells (1×10⁶ cells/mouse). After tumor formation, the mice were sacrificed and tumors were removed and their weights and volumes were calculated (Fig. 13A). The results showed that 25, 50 and 100 mg/kg bergamottin injection reduced the tumor weight from 1.61 g in the PBS-treated group (control) to 1.21, 0.42 and 0.15 g, respectively (Fig. 13B). Tumor weight in the nude mice was reduced much more significantly in the highest-concentration bergamottin group (100 mg/kg body weight) compared with the vehicle group (P<0.05). Likewise, 25, 50 and 100 mg/kg bergamottin injection reduced the tumor volume from 2.2 cm³ in the PBS-treated group (control) to 1.71, 1.1 and 0.51 cm³, respectively (Fig. 13C). The periodic measurement of the tumor xenograft volume indicated that the tumor volume in the nude mice was reduced considerably in the highest-concentration bergamottin group (100 mg/kg body weight) compared with the vehicle group (P<0.05).