Cedrol (>98%) was purchased from Chengdu Pufei De Biotech Co., Ltd. (Chengdu, China). N-acetyl-L-cysteine (NAC) and tocopherol, were purchased from Beyotime Biotech (Jiangsu, China). Specific antibodies against GAPDH (D16H11), microtubule associated protein light-chain 3 (LC3)B [(D11)-#3868], unc-51 like autophagy activating kinase 1 [ULK1; (D9D7)-#6439], p70S6K (#9204), phosphorylated (p-)mTOR (Ser2448), p-ULK1 (Ser317; (D2B6Y)12753), p-P70S6K (Thr421/Ser424) and secondary antibodies (anti-rabbit IgG, HRP-linked antibody; #7074) were purchased from Cell Signaling Technology (Danvers, MA, USA). RIPA lysis buffer was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 5-(6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (CM-DCFH₂-DA), propidium iodide (PI), trypsin and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 3-[4,5-Dimethyl-2-thiazolyl]-2,5-diphenyltetrazolium bromide (MTT), was purchased from Molecular Probes (Eugene, OR, USA). Fetal bovine serum (FBS), and penicillin-streptomycin were obtained from Hangzhou Sijiqing Biological Products Co., Ltd., (Hangzhou, China). Phospho-Akt [p-Akt, Ser-473; (D9E) #4060], phospho-PI3K (p-PI3K), PI3K, cytochrome c [(136F3) #4280] and Akt antibodies were purchased from Cell Signaling Technology. β-actin as well as secondary anti-mouse and anti-rabbit antibodies were purchased from Santa Cruz Biotechnology. Annexin V-FITC apoptosis detection kits and mitochondrial sensor kits containing JC-1 were purchased from Nanjing KeyGen Biotechnology Co., Ltd. (Nanjing, China).

The human NSCLC cell line A549 was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and was maintained at 37°C in a humidified atmosphere containing 5% CO₂. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich) supplemented with 5% FBS, 2% penicillin/streptomycin and 2% amphotericin B (Sigma-Aldrich).

The effects of cedrol on A549 cell viability were examined by performing an MTT assay. The cells (2×10⁵ cells/well in 100 µl medium) were seeded into 96-well plates for 24 h prior to drug treatment. Taxol (Sigma-Aldrich), which exhibits activity against a number of types of cancer cells, was used as a positive control. Following treatment with various concentrations of cedrol (0, 2.5, 5, 10, 20, 40 and 80 µM) for 24, 48 and 72 h, the cell plates were treated with MTT solution [10 µl; 5 mg/ml in phosphate-buffered saline (PBS) solution] for an additional 4 h at 37°C. The formazan crystals in the viable cells were solubilized with DMSO (150 µl) and the absorbance was measured on a microplate reader (ELX 800; Bio-Tek Instruments, Inc., Winooski, VT, USA) at a wavelength of 490 nm. The effects of cedrol on cell viability were calculated as an inhibition ratio (I%) using the following equation [optical density at 490 nm (OD₄₉₀)]: I % = [ OD 490 ( Control ) − OD 490 ( Treated ) ] OD 490 ( Control ) × 100

The A549 cells were seeded into 96-well plates and treated with cedrol for 24 h, and then 10 µl LDH release reagent was added to each well. After 1 h, the cell culture plate was centrifuged at 400 × g for 10 min and 120 µl supernatant was removed from each well and transferred into a new black 96-well plate. Next, 60 µl of LDH assay mixture was added to each well, and the plate was incubated at 37°C for 30 min. The absorbance was measured spectrophotometrically at a wavelength of 490nm.

The A549 cells were plated in 6-well plates at a density of 1×10⁵ cells/ml and then cultured for 24 h to facilitate total attachment to the surface of the plates. Subsequently, the cells were subjected to treatment with various concentrations of cedrol (0, 5, 20 or 40 µM) for 48 h. Following drug treatment, the culture plates were examined under an inverted light microscope (Nikon Corporation, Tokyo, Japan) and images were captured.

Intracellular ROS generation was evaluated using f luorescent CM-DCFH₂-DA. The A549 cells were seeded into 6-well plates and following adhesion, the cells were pre-treated with 10 µM CM-DCFH₂-DA for 20 min followed by co-incubation with various concentrations of cedrol for another 2 h and washed with ice-cold PBS twice. The cells were collected and analyzed using a flow cytometer (FACSCanto™; Becton-Dickinson, Franklin Lakes, NJ, USA) with wavelengths of excitation and emission at 488 and 525 nm, respectively. To explore the role of NAC and tocopherol in the cedrol-induced production of ROS, NAC (2.5 mM) and tocopherol (10 µM) were incubated with the A549 cells for 1 h prior to exposure to cedrol and ROS production was measured.

In order to confirm whether autophagy is induced by cedrol treatment in the A549 cancer cells, the cells were treated with cedrol at various concentrations (0, 5, 20 and 40 µM) for 24 h and the autophagosomes were visualized using TEM (TEM-1400 plus; JEOL USA, Inc., Peabody, MA, USA) as previously described [Zhou et al, 2014 (19)]. The cells were fixed in ice-cold 2.5% glutaraldehyde in PBS overnight at 4°C. The fixed samples were washed three times in PBS. After fixation, the samples were post-fixed in 1% osmium tetroxide containing 0.1% potassium ferricyanide for 1 h, and then subjected to TEM analysis.

The expression of various proteins in the cells was evaluated using western blot analysis. Briefly, after the cells were treated with cedrol, the total proteins were extracted with RIPA lysis buffer containing 1% cocktail and 1% phenylmethanesulfonylfluoride (PMSF). Protein concentrations were determined using a BCA protein assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Equivalent amounts of proteins were separated by 10–12% SDS-PAGE and transferred onto a PVDF membrane (Bio-Rad Laboratories, Hercules, CA, USA). After blocking with 5% nonfat milk in TBST (10 mM Tris-HCl, 500 mM NaCl, and 0.1% Tween-20) at room temperature for 1 h, the membrane was incubated with specific primary antibodies (1:1,000) overnight at 4°C. After washing with TBST, the membrane was incubated with the corresponding secondary antibodies (1:10,000). To probe for p-PI3K, Akt, p-Akt and β-actin, the membrane was incubated overnight at 4°C with the relevant antibodies. After washing with TBST three times, the membrane was incubated with alkaline phosphatase-conjugated goat anti-rabbit secondary antibodies at room temperature for 1 h followed by washing with TBST buffer three times. Signals were detected using an ECL Plus Chemiluminescence kit (Applygen Technologies, Inc., Beijing, China) on X-ray film. One hundred and seventy-three gray levels were analyzed using Gel-Pro 32 software (Media Cybernetics, Inc., Rockville, MD, USA).

Apoptosis was evaluated using an Annexin V-FITC apoptosis detection kit. Briefly, the A549 cells were seeded into 6-well plates, incubated overnight, and then treated with 5, 20 or 40 µM of cedrol for 48 h. The cells were harvested by trypsinization, washed twice with pre-chilled PBS (4°C) and centrifuged at 251 × g for 10 min. The cell pellet was resuspended in binding buffer (400 µl) and the rate of apoptosis was examined using Annexin V-FITC and PI double staining according to the manufacturer's instructions. After staining the cells with Annexin V-FITC/PI, flow cytometric analysis was performed using a FACSCalibur flow cytometer (Becton-Dickinson), and the data were analyzed using CellQuest software.

MTP was measured using JC-1 staining. Briefly, the A549 cells were seeded into 6-well plates and incubated without (control) or with (5, 20 or 40 µM) of cedrol for 48 h. After incubation, the cells were collected, and the cell pellets were washed twice with ice-cold PBS and then resuspended in DMEM at a density of 1×10⁶ cells/ml. The cells were then incubated with JC-1 (1 µl) at 37°C for 15 min. The stained cells were washed twice with PBS, resuspended in PBS (500 µl) and analyzed using flow cytometry with excitation and emission wavelengths of 488 and 530 nm, respectively. The fluorescence of the treated cells was compared with the untreated control group.

Data are presented as the means ± SEM of the control. All experiments were repeated at least three times. The differences between groups were analyzed by one-way ANOVA with Tukey's post hoc tests. P<0.05 and P<0.01 was considered to indicate a statistically significant difference.

The MTT assay was used to examine the inhibitory effects of cedrol on the proliferation of A549 cells. The chemical structure of cedrol is shown in Fig. 1A. The results indicated that cedrol markedly inhibited the proliferation of the human lung cancer cell line A549 (Fig. 1B). Furthermore, time- and concentration-dependent inhibition curves were observed in these cells. The calculated IC₅₀ values at 24, 48 and 72 h were 31.87±9.3, 14.54±2.18 and 5.02±0.7 µM), respectively. In addition, LDH release into the culture medium was also increased in a concentration-dependent manner (Fig. 2). The results of these two assays indicated that cedrol induced potent cytotoxic effects in the A549 cells in a dose-dependent manner.

The exposure of A549 cells to 5, 20 or 40 µM of cedrol for 48 h resulted in a substantial decrease in cell counts and furthermore, induced morphological changes that were characteristic of cytotoxicity (cell shrinking, loss of membrane integrity) in the A549 cells. The cells were examined under an inverted phase contrast microscope following exposure to cedrol (Fig. 3).

The effect of cedrol on intracellular ROS production was measured by flow cytometry using a fluorescent probe CM-DCFH₂-DA. As shown in Fig. 4A and B, ROS formation was markedly induced after treating the A549 cells with cedrol for 2 h. ROS generation occurred in a concentration-dependent manner and a two-fold increase in ROS production was observed following treatment with 20 µM cedrol. In addition, pre-treatment with NAC (2.5 mM) or tocopherol (10 µM) for 1 h almost completely reversed the cedrol-induced generation of ROS in the A549 cells (Fig. 5).

Cedrol-induced autophagy was first examined using TEM. TEM analysis revealed that compared with the control group (Fig. 6A), cedrol treatment (5, 20 and 40 µM) induced a large number of autophagosomes (Fig. 6B–D) and this number increased with increasing concentrations of cedrol. Cedrol-induced autophagy was also evaluated by western blot analysis. The results revealed that cedrol treatment induced a substantial increase in the protein expression of LC3B in the A549 cells in a dose-dependent manner (Fig. 7A). In addition, the results of western blot analysis indicated that cedrol treatment markedly suppressed the protein expression of p-mTOR and p-P70S6K and increased the protein expression of ULK1 and p-ULK1 in the A549 cells in a concentration-dependent manner (Fig. 7B).

To confirm whether cedrol-induced cell death in A549 cells may be mediated through the activation of the apoptotic cascade, the cells were treated with different concentrations of cedrol, stained with Annexin V-FITC/PI and analyzed for apoptosis using flow cytometry. We observed a dose-dependent increase in the percentage of apoptotic cells (2.02, 18.3, 29.61 and 53.41% following 48 h of treatment with 0, 5, 20 or 40 µM of cedrol, respectively) (Fig. 8). The results indicated that cedrol efficiently suppressed cell growth and induced apoptosis in human lung cancer cells in vitro.

The PI3K/Akt signaling pathway is recognized to be one of the key signaling pathways involved in the regulation of cell growth, proliferation and apoptosis. Akt is a major downstream target of PI3K, and Akt phosphorylation leads to the significant upregulation of Bcl-2 expression, and the downregulation of Bax expression. We examined the effects of cedrol on the PI3K/Akt signaling pathway by measuring the protein levels of p-PI3K, total Akt, and p-Akt. It was observed that cedrol significantly downregulated the expression of p-Akt and p-PI3K in the A549 cells in a dose-dependent manner whereas the total Akt protein levels remained constant during all treatments (Fig. 9).

Mitochondria are important organelles that are involved in the release of apoptotic signals during an intrinsic pathway for the execution of apoptosis (20). Mitochondrial dysfunction leads to a reduction of the MTP as well as the release of cytochrome c from the mitochondria into the cytosol (21), which is one of the mechanisms through which caspase activation induces apoptotic cell death. To determine the effects of cedrol on MTP, the A549 cells were treated with cedrol for 48 h, and MTP was measured using JC-1 staining in conjunction with flow cytometry. This assay is based on the principle that the red fluorescent JC-1 dimers will be present in areas with high MTP (upper-right quadrant of the fluorescence cytogram), whereas green fluorescent JC-1 monomers will be prevalent in areas with low MTP (lower-right quadrant of the fluorescence cytogram). As shown in Fig. 10, green fluorescence increased from 0.25 to 55.92% as the concentration of cedrol increased. The results indicated that cedrol-induced apoptosis was caused by mitochondrial dysfunction induced by a reduction in MTP.