Human SCLC Calu-6 cells and NSCLC adenocarcinoma A549 cells were obtained from the American Type Culture Collection (ATCC). These cell lines were maintained in a standard humidified incubator containing 5% CO₂ at 37°C. The lung cancer cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) (Sigma-Aldrich; Merck KGaA) and 1% penicillin-streptomycin (Gibco BRL; Thermo Fisher Scientific, Inc.). Cells were grown in BD Falcon 100-mm plastic cell culture dishes (BD Biosciences) and harvested with trypsin-EDTA (Gibco BRL; Thermo Fisher Scientific, Inc.). Exponentially growing cells were used for the experiments.

PG was acquired from Sigma-Aldrich Co (Merck KGaA). PG was dissolved in ethanol at 200 mM as a stock solution. Ethanol (0.2%) was used as a control vehicle and did not influence cell growth or cell death. Stock solution was wrapped in foil and kept at 4°C or −20°C.

The influence of PG on the growth of lung cancer cells was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich; Merck KGaA) assays. Briefly, 5×10⁴ cells were seeded into each well of 96-well microtiter plates (Nunc). After incubation with the designated doses of PG for 24 or 72 h, 20 µl of MTT solution [2 mg/ml in phosphate-buffered saline (PBS); Gibco BRL; Thermo Fisher Scientific, Inc.] was added to each well. The plates were incubated for 4 h at 37°C. The medium in plates was removed via pipetting, and 100–200 µl of DMSO was added to each well to solubilize formazan crystals. Optical density was measured at 570 nm with a microplate reader (Synergy™ 2, BioTekR Instruments Inc.). Each plate contained multiple wells at a given experimental condition and multiple control wells. This procedure was replicated for 2 to 4 plates per condition.

Cell cycle and sub-G1 distributions in cells were determined using propidium iodide (PI, Sigma-Aldrich; Merck KGaA; Ex/Em = 488 nm/617 nm) staining. Briefly, 1×10⁶ cells in BD Falcon 60-mm culture dishes (BD Biosciences) were incubated with the designated concentrations of PG for 24 or 72 h. Cells were washed twice with PBS and fixed in cold 70% ethanol. Cells were again washed with PBS, and then incubated with 10 µg/ml PI concurrently with RNase (Sigma-Aldrich; Merck KGaA) at a concentration of 5×10⁵ cells/ml in PBL at 37°C for 30 min. The proportions of cells in different phases of the cell cycle or having sub-G1 DNA content were measured and analyzed with a FACStar flow cytometer (BD Sciences).

Apoptosis was identified via Annexin V-fluorescein isothiocyanate staining (FITC, Thermo Fisher Scientific, Inc.; Ex/Em = 488/519 nm). Annexin V-FITC is used to detect phosphatidylserine exposing cells thereby marking apoptotic cells. Briefly, 1×10⁶ cells in BD Falcon 60-mm culture dishes (BD Biosciences) were incubated with the designated concentrations of PG for 24 or 72 h. Cells were washed twice with cold PBS and then suspended in 200 µl of binding buffer (10 mM HEPES/NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂) at a concentration of 5×10⁵ cells/ml at 37°C for 30 min. Annexin V-FITC (2 µl) was added to the solution, and cells were analyzed with a FACStar flow cytometer (BD Sciences).

The MMP (ΔΨm) was monitored using a Rhodamine 123 cationic fluorescent dye (Sigma-Aldrich; Merck KGaA; Ex/Em = 485/535 nm), which preferentially enters into mitochondria with high MMP (∆Ψm). Depolarization of MMP (∆Ψm) results in the loss of Rhodamine 123 from the mitochondria and reduces the intracellular fluorescence intensity of this dye. In brief, 1×10⁶ cells in 60-mm culture dishes (Nunc) were incubated with the indicated doses of PG for 24 h. Cells were washed twice with PBS and incubated with Rhodamine 123 (0.1 mg/ml) at a concentration of 5×10⁵ cells/ml in PBL at 37°C for 30 min. Rhodamine 123 staining intensities were determined using a FACStar flow cytometer (BD Sciences). Rhodamine 123 negative (−) cells indicated the loss of MMP (∆Ψm) in the lung cancer cells. MMP (∆Ψ_m) levels in cells, except MMP (∆Ψ_m) loss cells, were expressed as proportions compared with the control cells.

Protein expression levels were evaluated via western blotting. Briefly, 5×10⁶ cells in BD Falcon 100-mm culture dishes (BD Biosciences) were incubated with the indicated concentrations of PG for 24 h. Cells were washed with PBS and 4 volumes of lysis buffer (Intron Biotechnology) was added. Total proteins (30 µg) were resolved via 8–15% SDS-PAGE gels and then transferred to Immobilon-P PVDF membranes (Millipore) by electroblotting. Membranes were probed with anti-Bcl-2 (cat. no. 2872, 1:1,000 dilution), anti-caspase-3 (cat. no. 9662, 1:1,000 dilution), anti-PARP (cat. no. 9542, 1:1,000 dilution), anti-cleaved PARP (cat. no. 9541, 1:1,000 dilution) antibodies (Cell Signaling Technology, Inc.), and anti-β-actin antibody (sc-81178, 1:1,000 dilution, Santa Cruz Biotechnology, Inc.). Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (sc-2004 or sc-2005, 1:1,000 dilution, Santa Cruz Biotechnology, Inc.). Western blots were developed using an EZ-Western Lumi Pico ECL solution kit (DoGen, Korea).

The activities of caspase-3 and caspase-8 were evaluated using Caspase-3 and Caspase-8 Colorimetric Assay Kits (R&D Systems, Inc.). In brief, 1×10⁶ cells were incubated with the specified concentrations of PG for 24 h. Cells were washed with PBS and 4 volumes of lysis buffer (Intron Biotechnology) was added. Samples containing 50 µg of total protein were added to 2X Reaction buffer containing DEVD-pNA for caspase-3 activity or IETD-pNA for caspase-8 activity in 96-well microtiter plates (Nunc) and incubated at 37°C for 1 h. Optical density was measured at 405 nm using a microplate reader (Synergy™ 2). Each plate contained multiple wells of a given experimental condition as well as multiple control wells. Caspase activity is expressed in arbitrary absorbance units (absorbance at a wavelength of 405 nm).

The results represent the mean of at least three independent experiments (mean ± SD). Data were analyzed using Instat software (GraphPad Prism 5.0; GraphPad Software, Inc.). One-way analysis of variance with post hoc analysis using Tukey's multiple comparison test was applied to judge statistical significance which was defined at P<0.05.

The effect of PG on the growth of Calu-6 and A549 lung cancer cell types was observed using MTT assays. A dose-dependent reduction in cell growth was observed in Calu-6 cells with a half maximal inhibitory concentration (IC₅₀) of approximately 800 µM following treatment with PG for 24 h (Fig. 1A). Additionally, a 50 µM concentration of PG appeared to significantly reduce the growth of Calu-6 cells by approximately 60% at 72 h (Fig. 1A). Thus, the IC₅₀ of PG in Calu-6 cells at 72 h was less than 50 µM. The growth of A549 cells was also reduced with an IC₅₀ of ~800 µM after a 24-h incubation with PG (Fig. 1B). In addition, the IC₅₀ of PG in A549 cells at 48 h was approximately 150 µM (data not shown). Treatment with 100 µM PG decreased the growth of A549 cells by approximately 50% at 72 h (Fig. 1B).

Because the growth inhibition of Calu-6 and A549 cells by PG could be explained by an arrest during cell cycle progression, allocations of cells in different stages of the cell cycle were observed after a 24- or 72-h incubation period with PG. DNA flow cytometric analysis indicated that tested doses of PG induced a G1 phase arrest of the cell cycle in Calu-6 cells at 24 and 72 h (Fig. 2A and B). Particularly, concentrations of 200 and 400 µM PG showed a significant increase in the G1 phase at 24 h (Fig. 2A and B). In addition, the tested doses of PG induced a G1 phase arrest of the cell cycle in A549 cells at 24 h (Fig. 2C and D). Treatment with 100 and 200 µM PG also increased the proportion of cells at the G1 phase at 72 h (Fig. 2C and D).

Whether PG induces cell death was evaluated using sub-G1 cells and Annexin V-staining cells. As shown in Fig. 3A, the tested doses of PG increased the quantity of sub-G1 cells in Calu-6 cells at 24 or 72 h, but the effects were not dose-dependent. PG also increased the quantity of sub-G1 cells in A549 cells at 24 or 72 h (Fig. 3B). Furthermore, 100–400 µM PG did not increase the percentage of Annexin V-stained cells in both Calu-6 (Fig. 4A and B) and A549 cell lines (Fig. 4A and C) and 800 µM PG slightly increased the percentage of Annexin V-staining cells in these cells (Fig. 4). However, the percentage of Annexin V-stained cells in Calu-6 and A549 cells was significantly increased after treatment with 1,600 µM PG at 24 h (Fig. 4).

As apoptosis is closely related to the collapse of MMP (∆Ψm), loss of MMP (∆Ψm) in PG-treated cells was evaluated using Rhodamine 123 dye. Loss of MMP (∆Ψm) in both lung cancer cell types was dose-dependently induced by PG at concentrations of 100–1,600 µM at 24 h (Fig. 5A-C). After exposure to 800 µM PG, the proportions of cells with MMP (∆Ψ_m) loss in Calu-6 and A549 cell lines were approximately 30 and 45%, respectively (Fig. 5A-C). In relation to the levels of MMP (∆Ψ_m) in both lung cancer cell lines at 24 h, 100 µM PG increased the MMP (∆Ψ_m) level in Calu-6 cells whereas PG at 200–1,600 µM significantly decreased the MMP (∆Ψ_m) level in these cells (Fig. 5A and D). Treatment with 100–1,600 µM PG also reduced MMP (∆Ψ_m) levels in A549 cells (Fig. 5A and E). The levels of MMP (∆Ψ_m) were approximately 30 and 20% in Calu-6 and A549 cell lines treated with 800 µM PG, respectively (Figs. 5A, D and E).

Since PG induced cell death in both lung cancer cell types, the levels of apoptosis-related proteins were assessed by western blot analysis. Examination of Bcl-2 regulation in PG-treated cells revealed that Bcl-2 protein levels were reduced following treatment with 200–800 µM PG in both lung cancer cell types (Fig. 6A and B). Caspase-3 plays an essential role as an executioner in apoptosis (25). Whether PG activates caspase-3 in PG-treated lung cancer cells was also examined. Levels of procaspase-3 (32 kDa precursor) were visibly reduced following treatment with 400–800 µM PG in the Calu-6 cells (Fig. 6A) and following treatment with 200–800 µM PG in the A549 cells (Fig. 6B). Cleavage of PARP provides one of the most recognizable markers of apoptosis (26). While the intact 116 kDa moiety of PARP was reduced in the 400–800 µM PG-treated Calu6 cells, the cleaved form of PARP increased in these cells (Fig. 6A). PG at 200–800 µM also reduced the intact form of PARP in the A549 cells (Fig. 6B). Furthermore, 400–800 µM PG treatment significantly increased caspase-3 activities in the Calu-6 cells (Fig. 6C). Caspase-8, which is correlated with the cell death receptor pathway (25), was also significantly activated by treatment with 800 µM PG in Calu-6 cells (Fig. 6C). However, 400 µM PG downregulated the activity of caspase-8, compared with basal activity of the control cells (Fig. 6C).