Oxymatrine was a kind gift from Professor Min Liu, (The Xiang'an Hospital of Xiamen University, Xiamen, China). It was dissolved in PBS solution (stock concentration of 100 mg/ml). RPMI-1640 medium and newborn calf serum (NCS; Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) were purchased from Beijing DingguoChangsheng Biotechnology, Co. Ltd. (Beijing, China). A549 cells were cultured in 1640 medium supplemented with 10% calf serum and 1% garamycin at 37°C in an incubator in an environment containing 5% CO₂.

A549 cells were seeded into 12-well culture plates (Corning Incorporated, Corning, NY, USA) at a density of 1×10⁵ cells per well by using Blood Counting chambers, that would allow the formation of a 90% monolayer following 24 h. A total of six different final concentrations of oxymatrine (0.1, 0.5, 1.0, 1.5, 2.0 and 2.5 mg/ml) were determined and added into the wells. The treated cells were then incubated at 37°C for different time periods. Pictures were taken by light microscope to detect cell morphological alterations at different time points in oxymatrine-treated A549 cells.

A549 cells were incubated in 96-well plates (1×10⁴ cells/well) in an incubator containing 5% CO₂ at 37°C. Then, cells were treated with oxymatrine at different final concentrations for 24 h. A total of 20 µl MTT [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide, stock 0.1 M] was added into each well and cells were placed in the dark for 4 h at 37°C. Following aspiration of supernatant, dimethyl sulfoxide (100 µl/well) was added to the wells, and cells were kept in the dark at room temperature, on a mixer for 30 min. The absorbance (OD₅₇₀) was measured at a wavelength of 495 nm by a microplate spectrophotometer (BioTek China, Beijing, China) to determine cell viability. All the experiments were repeated in triplicate. P<0.05 and P<0.01 were considered to indicate a statistically significant difference.

A549 cells were incubated in 6-well plates (3×10⁵ cells/well) and incubated in an environment containing 5% CO₂. Oxymatrine (1.5 mg/ml) was used to treat cells in exponential growth period for 24, 48 and 72 h, respectively. Following immobilization with 4% paraformaldehyde for 10 min at room temperature, A549 cells were stained with Hoechest 33342 (10 µg/ml) and PI, (10 µg/ml) for 10 min in 37°C incubator successively. Following washing three times with PBS, the treated cells were sealed with glycerol (50% in PBS) and then observed under the fluorescence microscope (Nikon TE2000-U or Nikon 80i; Nikon Corporation, Tokyo, Japan).

For the mitochondrial membrane potential assay, the green JC-1 (GenView SA, Lausanne, Switzerland) signals were measured at Ex 485 nm/Em 528 nm, whereas the red signals were detected at Ex 540 nm/Em 590 nm. The absorbance was determined by a microplate spectrophotometer (BioTek China).

For caspase inhibition analysis, A549 cells were firstly incubated with 50 µM z-VAD-FMK (Beyotime Institute of Biotechnology, Haimen, China) for 30 min. Then, the A549 cells were treated with oxymatrine (1.5 mg/ml) for another 48 h. Finally, the fluorescence microscope analysis was carried out in accordance with the aforementioned procedure.

A549 cells (3×10⁵ cells/well) were cultured under the aforementioned conditions, and oxymatrine (final concentration of 1.5 mg/ml) was incubated with cells for 48 h. Then, apoptotic cells were analyzed according to the TUNEL kit manufacturer's protocol (DeadEND^™ Colorimetric Apoptosis Detection System; Promega Corporation, Madison, WI, USA).

A549 cells (3×10⁵ cells/ml) were seeded into a 30-mm cell culture dish and treated with oxymatrine (1 mM) for 24, 48 and 72 h, respectively. The A549 cells were fixed (70% ethanol) for 2 h at 4°C, washed with PBS, permeabilized (0.1% Triton X-100) and stained (PI-FITC) in accordance with the procedure described in Zhou et al (17). Then, cell samples were employed for the flow cytometry assay. Data were collected and analyzed by the CellQuest Prosoftware (version 6.0; FACS Calibur; BD Biosciences, Franklin Lakes, NJ, USA).

Mock-treated or oxymatrine-treated (1.5 mg/ml) A549 cells (48 h) were harvested by scraping, washed with PBS, frozen for 30 min and centrifuged (16,000 × g for 15 min at 4°C). The supernatants were assayed by a plate reader (Model 550 Microplate Reader; Bio-Rad Laboratories, Inc., Hercules, CA) at a wavelength of 405 nm, for the activities of caspase-3, 8, 9 in the presence of 20 µM Ac-DEVD-pNA, Ac-IETD-pNA and Ac-LEHD-pNA (Beyotime Institute of Biotechnology, Haimen, China), respectively. The experiments were carried out in triplicate.

The migratory activity of A549 cells was determined using a wound healing assay, as previously described (17). Briefly, one pipette tip was used to make a scratch wound across each well in a 12-well plate. Following washing and removing any loosely held cells, oxymatrine (1.5 mg/ml) was added into each well for 24, 48 and 72 h, respectively. An inverted microscope was used to track the migration process of A549 cells.

One-way analysis of variance followed by LSD and Tukey post-hoc tests was used to evaluate differences between groups. All data were analyzed by SPSS software (version 19.0; IBM Corp., Armonk, NY, USA) and expressed as the mean ± standard error. P<0.05 was considered to indicate a statistically significant difference.

Microscope observation analysis indicated that A549 cells became rounded and granular following treatment with oxymatrine at different concentrations (Fig. 1). At 24 h, the treated cells did not appear to exhibit any properties characteristic of apoptosis. However, over the increasing time period, the treated A549 cells detached from the monolayer and floated in the cell medium, and this effect appeared to be enhanced with increased concentrations of oxymatrine.

Furthermore, MTT analysis was applied to identify the A549 cell viability following treatment. The experiments demonstrated that oxymatrine inhibited A549 cell growth in a time- and dose-dependent manner (Fig. 2). When the treatment concentration of oxymatrine increased, the A549 cell viability continuously decreased. Overall, the aforementioned results suggested that oxymatrine had an anti-proliferative effect on the A549 lung cancer cells.

To further identify the apoptosis mechanism of A549 induced by oxymatrine, Hoechst/PI dual-staining assay was employed to detect possible cell nuclei alterations. Generally, Hoechst enters into the normal cytomembrane and stains the nucleolus blue, whereas PI is a nucleic acid dye that only has access to the damaged cytomembrane and stains nuclei red, therefore is often used for assessment of the late apoptotic cells. Hoechst 33342 staining exhibited apparent nuclear fragmentation at 48 h (Fig. 3A), whereas the nuclei of control cells remained intact. The red fluorescence of PI was additionally detected, suggesting that oxymatrine induce apoptosis of A549 cells.

Given that apoptosis is often caspase-dependent, the caspase inhibitor z-VAD-FMK was used to pre-incubate the A549 cells, and then fluorescence observation analysis revealed that z-VAD-FMK, in part, inhibited the fragmentation of A549 cell nuclei (Fig. 3B). This suggested that the oxymatrine-induced apoptosis of A549 cells was caspase-dependent.

Fluorescence microscopy analysis was used to study apoptosis of A549 cells following oxymatrine treatment. In addition to this, TUNEL and flow cytometry analysis were applied to further verify the apoptotic characteristics of A549 cells. TUNEL analysis demonstrated that the apoptotic cells contracted and the nuclei appeared navy and the perinuclei brown in the oxymatrine-treated group, whereas the positive signals were not detected in control cells (Fig. 4).

Furthermore, as a lipophilic and cationic dye, JC-1 aggregates locate in areas with high mitochondrial membrane potential (∆Ψm) and emits red fluorescence, and JC-1 monomers emit green fluorescence, and locate in low ∆Ψm cell areas. The ratio of red/green fluorescence acts as a marker of ∆Ψm alterations in cells (18). In the present study, JC-1 staining revealed a decrease of red/green ratio following treatment with oxymatrine for 12, 48 and 72 h, respectively (Fig. 5A). The decrease of ∆Ψm further verified that oxymatrine induced A549 cell apoptosis.

In addition, to determine the cause of apoptosis, flow cytometry analysis was applied to study the cell arrest of oxymatrine-treated A549 cells. The results suggested that the DNA synthesis decreased in a time dependent manner, and the number of G0/G1 cells increased prior to 48 h (Fig. 5B). Compared with control cells, treated groups at different time-points all revealed S phase arrest. The above mentioned experiments verified that oxymatrine resulted in A549 cell arrest and induced apoptosis. Future studies should focus on the specific molecular apoptotic mechanism of A549 cells.

To further investigate whether the apoptosis induced by oxymatrine was caspase-dependent or not, the oxymatrine-treated (48 and 72 h) or untreated control A549 cells were harvested by scraping. The supernatants of these cell lysates were detected for the activities of several key caspases in the process of apoptosis. The results demonstrated that the absorbance (OD_405nm, n=3) of oxymatrine-treated cell supernatants all increased compared with the control cells (P<0.05; Fig. 6) which suggested that caspases-3, 8 and 9 were activated following oxymatrine treatment in A549 cells, and the induced apoptosis was caspase-dependent.

Due to their proliferative abilities, a further property of malignant tumor cells is the ability to migrate (19). The present study performed the wounding healing assay to examine the anti-migratory abilities of oxymatrine. The results demonstrated that following treatment with oxymatrine (1.5 and 2.0 mg/ml) for different time periods, a decreased number of A549 cells migrated to the blank field (Fig. 7A). Increased concentrations of oxymatrine resulted in a smaller migrated cell number ratio (Fig. 7B). This suggested that oxymatrine inhibited migration of A549 cells. Future investigations should focus on factors involved in the migration of A549 cells following oxymatrine administration.