RPMI-1640, fetal bovine serum (FBS), penicillin and streptomycin were purchased from HyClone Laboratories Inc., (Logan, UT, USA). Bovine serum albumin (BSA), 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), 4,6-diamidino −2-phenylindole (DAPI) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). TIMP-1 (mouse, monoclonal, sc-365905), Cdk2 (mouse, monoclonal, sc-6248), cyclin A (rabbit, polyclonal, sc-751), cyclin E (rabbit, polyclonal, sc-198), P-GSK3β (mouse, monoclonal, sc-81495), GSK3β (mouse, monoclonal, sc-81462), PI3K (rabbit, polyclonal, sc-602) and β-actin (mouse, monoclonal, sc-47778) were all purchased from Santa Cruz Biotechnology Inc., (Santa Cruz, CA, USA). Cleaved PARP (rabbit, polyclonal, #9541s), survivin (rabbit, monoclonal, #2808s), Cdk4 (rabbit, monoclonal, #12790s), cyclin D (rabbit, monoclonal, #2978s), p-Akt (rabbit, monoclonal, #4058s) and Akt (rabbit, polyclonal, #9272s) were purchased from Cell Signaling Technology (Beverly, MA, USA). Caspase-3 (active, rabbit, polyclonal, ALX-210-807-c100), caspase-9 (active, rabbit, polyclonal, ALX-210-816-c100), cIAP-1 (rat, monoclonal, ALX-803-335-c100) and cIAP-2 (rat, monoclonal, ALX-803-341-c100) were purchased from Enzo Life Sciences (Farmingdale, NY, USA) and diluted 1:1,000. Primary antibodies were diluted 1:1,000. Peroxidase-conjugated secondary antibodies (rabbit, monoclonal, sc-2357; mouse, polyclonal, sc-2005; rat, polyclonal sc-2006; dilution 1:2,000) were purchased from Santa Cruz Biotechnology. An enhanced chemiluminescent (ECL) kit was obtained from Amersham Pharmacia Biotech (Buckinghamshire, UK).

For the preparation of PGD dried roots, P. grandiflorum (5 kg) underwent extraction three times with methanol at room temperature for seven days. Concentration of the solvent gave a brown syrupy extract (1.4 kg) which was suspended in water and then partitioned successively with ethyl acetate (63 g) and n-butanol (130 g). The n-butanol layer was suspended in H₂O (2 liters) and poured into a Diaion HP-20 column (Φ = 5.0×100 cm; Mitsubishi Chemicals Corp., Tokyo, Japan), which was stabilized with H₂O. The column was washed with H₂O (2 liters) and then eluted with MeOH (5 liters). Polygalacin D2 (PD2) was prepared from the extract of P. grandiflorum for experimental procedures using our method (15).

Human lung cancer cells NCI-A549 and H460 were obtained from the Korean Cell Line Bank (Seoul, Korea) and grown in RPMI-1640 containing 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. BEAS-2B, a human bronchial epithelial cell line, was purchased from the American Type Culture Collection (CRL-9609; ATCC, Manassas, VA, USA) and maintained in bronchial epithelial cell basal medium (BEBM) containing 1% penicillin/streptomycin. The cell lines were maintained in a humidified incubator, with an atmosphere of 5% CO₂ at 37°C. The effect of PGD on cell proliferation was determined using an MTS assay. The cells (2×10³/well) were seeded in 96-well culture plates and incubated for 24 h. Various concentrations (0–40 µM) of PGD were added and the cells were incubated for 48 h. The optical density (OD) of each culture well was assessed at 490 nm using a microplate reader (Titertek Multiskan; Flow Laboratories, North Ryde, NSW, Australia). Cell proliferation was calculated using the following formula: (mean absorbance value of treated cells/mean absorbance value of untreated cells) × 100.

Cell nuclear morphology was assessed by fluorescence microscopy following DAPI staining. A549 and H460 cells were treated with PGD for 48 h. The cells were washed with phosphate-buffered saline (PBS), fixed with ice cold 70% ethanol, stained with DAPI and incubated for 5 min at 37°C, in the dark. The cells were then washed with PBS and visualized using a fluorescence microscope (Carl Zeiss AG, Oberkochen, Germany).

The degree of apoptosis was assessed using Muse cell analyzer (EMD Millipore, Billerica, MA, USA) according to the manufacturers instructions. A549 and H460 cells were seeded in 6-well plates (10⁵ cells/well) and treated with PGD (0, 10, 20 and 40 µM) for 48 h. The harvested cells were washed with 1 ml of PBS, resuspended in 150 µl FBS free media and 150 µl of the Muse™ Annexin V Dead Cell kit (EMD Millipore) was added. Then, the cells were incubated at room temperature (RT) for 20 min in the dark. The samples were assessed with the Muse cell analyzer (EMD Millipore).

A549 and H460 cells were seeded in 6-well plates (10⁵ cells/well) and treated with PGD (0, 10, 20 and 40 µM) for 48 h. The harvested cells were washed with 1 ml PBS and fixed with ice-cold 70% EtOH over 3 h. After fixation, the cells were washed with PBS and suspended in 200 µl PBS. Muse™ Cell cycle reagent (200 µl; EMD Millipore) was then added and the cells were incubated at RT for 30 min in the dark. The samples were assessed with the Muse cell analyzer (Merck Millipore).

Cell extracts were subjected to SDS gel electrophoresis and then transferred onto a polyvinylidene fluoride membrane (PVDF; EMD Millipore). The membrane was blocked with 5% skim milk for 1 h and probed with primary antibodies. After a series of washes, the membrane was further incubated with a secondary antibody conjugated with horseradish peroxidase (HRP). The proteins were then supplemented with ECL prime western blotting detection reagents (GE Healthcare Life Sciences, Little Chalfont, UK). The bands were evaluated using the ImageQuant LAS 4000 mini biomolecular imager (GE Healthcare Life Sciences).

The statistical analysis was performed using one-way analysis of variance (ANOVA), followed by Scheffes test for multiple comparisons. Data are presented as the means ± standard deviations (n=5). All calculations were performed using SPSS statistics 23 software (SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

MTS assays were performed to determine the effects of PGD (Fig. 1) on the proliferation of lung cancer cell lines. Cancer cells were treated with increasing concentrations of PGD. As shown in Fig. 2A, PGD inhibited the growth of the treated cell lines in a dose-dependent manner. The IC₅₀ concentrations of PGD were 26.49±1.45 µM in the A549 cells and 20.52±0.30 µM in the H460 cells. Treatment with PGD at 40 µM reduced cell viability below 70% in normal airway bronchus cell line (Fig. 2B). Additional experiments were performed using PGD under 20 µM.

To determine whether the decreased viability of A549 and H460 cells occurs as a result of apoptosis, we investigated the apoptotic effects of PGD in NSCLC cell lines. In the present study, pemetrexed (PTX) was used as a control reagent to assess the apoptotic effects of PGD. PTX has been widely used in the clinical treatment of non-small cell lung cancer. As displayed in Fig. 3C and F, after treatment with PGD, nuclear condensation (white arrows) was observed in A549 and H460 cells. In addition, treatment with PGD increased the proportion of early and late apoptotic cells (Fig. 3A, B, D and E). Collectively, PGD-inhibited proliferation of NSCLC cells was due to cell apoptosis.

To elucidate the mechanism by which PGD induced apoptosis, the expression levels of proteins involved in the apoptosis pathway were examined using western blot analysis. The results indicated that treatment with PGD induced the cleavage of caspase-3 and −9, as well as PARP in both A549 and H460 cells (in lanes 2 and 3 compared with lane 1; Fig. 4). The expression levels of cleaved caspase-3 and −9, as well as PARP were markedly increased when compared with those in non-treated cells.

To further investigate the effects of PGD on the expression of the IAP family of proteins which play a significant role in intrinsic programmed cell death, NSCLC cells were treated with various concentrations of PGD and then, the expression levels of the IAP family of proteins including survivin, c-IAP-1 and c-IAP-2 were determined using a western blot assay. The expression of survivin, c-IAP-1 and c-IAP-2 was decreased, notably at the highest concentration of PGD, compared with that in non-treated cells. The expression of c-IAP-2 in A549 cells and the expression of survivin and c-IAP-2 in H460 cells was decreased to undetectable levels (Fig. 5). As a result, PGD may exert its apoptotic effects by regulating the apoptotic proteins and the IAP family of proteins.

To determine whether the effects of PGD on the inhibition of NSCLC cell-proliferation were related to cell-cycle arrest, the number of cells in the G0/G1, S and G2/M phases were assessed by flow cytometry. Treatment with PGD inhibited the progression of cell cycle in A549 and H460 cells. As displayed in Fig. 6, the percentage of cells in the G0/G1 phase was significantly increased in PGD-treated A549 and H460 cells compared with non-treated cells. Furthermore, treatment with PGD significantly increased the percentages of the sub G0 phase in both cell lines. In addition, PGD decreased the percentage of cells in the S and G2/M phases.

To evaluate the underlying mechanism of the effects of the cell cycle-arrest of PGD, the expression levels of proteins involved in cell cycle progression were determined using western blot analysis. As displayed in Fig. 7, PGD reduced the expression levels of proteins related to the progression of the cell cycle such as TIMP-1, Cdk2, Cdk4, cyclin A, D and E in both cell lines. In addition, PGD reduced the phosphorylation of GSK3β (Fig. 8), which is also involved in cell cycle progression. These results indicated that the inhibitory effects of PGD on cell growth via cell cycle arrest were induced as a result of a decrease in the expression of proteins related to cell cycle progression.

Previous research reported that the PI3K/Akt signaling pathway plays a critical role in cell survival. To confirm whether the inhibitory activity of PGD on cell growth was related to the PI3K/Akt signaling pathway, western blot analysis was performed. Treatment with PGD reduced the phosphorylation of Akt, while total Akt remained the same (Fig. 8). In addition, the expression of PI3K was reduced at the highest concentration of PGD compared with the non-treated group. In addition, as displayed in Fig. 9, co-treatment with LY294002 (the inhibitor of PI3K) decreased the expression of survivin and enhanced cleavage of PARP.