Human SPC-A-1 lung cancer cells (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were cultured in Dulbecco's modified Eagle medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (Sangon Biotech Co., Ltd., Shanghai, China). Cells were maintained in a humidified atmosphere of 5% CO₂ at 37°C. Fan (purity, >98.0%; Nature Standard Ltd., Shanghai, China) was prepared as a 50 mM stock solution in dimethyl sulfoxide (DMSO), prior to supplementation into the medium at various concentrations, for 48 or 72 h.

Cells were grown in 96-well culture plates and treated with various dosages of Fan (1.25, 2.5, 5, 10, 20 and 40 µM), as required, prior to incubation with 10 µl CCK-8 for 2 h. Following this, a Model 550 microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to measure the optical density (OD) of the samples at a wavelength of 450 nm. The cell inhibitory rate (IR) was calculated, as follows: IR = [1 - (OD_experiment - OD_blank) / (OD_control - OD _blank)] × 100%.

Following treatment with Fan, phase contrast imaging and Giemsa staining assays were used to analyze the proliferation of SPC-A-1 lung cancer cells. SPC-A-1 cells were treated with various concentrations of Fan (0, 2.5, 5 and 10 µM) and, after 48 h, the cells were visualized under an inverted microscope (CKX41; Olympus, Tokyo, Japan) prior to staining with a Giemsa assay (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer's instructions. In brief, the cells were fixed with the included solution I for 1 min and then solution II was added to stain the cells for another 5 min. Subsequently, the solution was removed and the images of cells were obtained using the Olympus CKX41 microscope.

SPC-A-1 cells were cultivated in a 6-well plate for 24 h, prior to treatment with Fan (0, 2.5, 5 or 10 µM) or equal volumes of DMSO. Following 48 h incubation, the cells were collected, fixed in 70% ice-cold ethanol (Sangon Biotech Co., Ltd.) and maintained at 4°C overnight. Cells were then washed in phosphate-buffered saline and the resultant pellet was re-suspended in 200 µg/ml RNase (Sangon Biotech, Co. Ltd.) for 1 h at 37°C. Cells were subsequently stained with 50 µg/ml propidium iodide, and analyzed using a flow cytometer (FACSCalibur; Beckman Coulter, Inc., Fullerton, CA, USA).

SPC-A-1 cells were treated with various concentrations of Fan for 48 h and the mRNA expression levels of genes that regulate the cell cycle were examined. Cells were collected and total RNA was extracted using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA synthesis was performed using a RevertAid™ First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.) with 3 µg total RNA, random hexamers (Fermentas; Thermo Fisher Scientific, Inc.) and specific oligonucleotide primers to detect the expression levels of cyclin D1, CDK4 and CDK6 mRNA. The sequences of the primer pairs were as follows: Cyclin D1, forward 5′-ATGCTGGAGGTCTGCGAGGA-3′ and reverse 5′-TTCGATCTGCTCCTGGCAGG-3′; CDK4, forward 5′-TGGCTTTACTGAGGCGACTG-3′ and reverse 5′-ACGGGTGTAGTGCCATCTG-3′; CDK6, forward 5′-GGAGTGCCCACTGAAACCAT-3′ and reverse 5′-GTGAGACAGGGCACTGTAGG-3′; and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), forward 5′-GAGAAGGCTGGGGCTCATTT-3′ and reverse 5′-GTCAGGTCCACCACTGACAC-3′. GAPDH was used as an internal control. PCR was performed at a final reaction volume of 25 µl, containing 1 µl cDNA, 1.5 mM MgCl₂, 1 U Taq DNA polymerase, 0.2 mM dNTP and 20 pM of each gene-specific oligonucleotide primer. The PCR reaction conditions were as follows: Denaturation at 94°C for 30 sec, annealing at 52–56°C for 30 sec, and extension at 72°C for 45 sec. The amplified products were run on 1.5% agarose gel and documented using a Gel Doc XR+ system (Bio-Rad Laboratories, Inc.). The densitometric analysis of the RT-qPCR results was performed using Quantity One software, version 4.6.0 (Bio-Rad Laboratories, Inc.) using GAPDH for normalization.

SPC-A-1 cells were cultivated in 6-well plates for 24 h, prior to treatment with Fan (0, 2.5, 5 and 10 µM) or DMSO (0.02%) for 48 h. Protein expression was detected using 10% SDS-PAGE at 250 V for 90 min. Subsequently, 20–30 µg total protein was transferred to polyvinylidene difluoride membranes and the membranes were blocked for 60 min with freshly prepared 5% non-fat milk in Tris-buffered saline and Tween-20 (TBST). Following this, the membranes were incubated with rabbit monoclonal pRb (1:1,500; #8180), polyclonal E2F-1 (1:2,000; #3742) and GAPDH (1:4,000; #5174; Cell Signaling Technology, Inc., Danvers, MA, USA) primary antibodies, washed three times with TBST, and incubated with goat anti-mouse or goat anti-rabbit IgG-horseradish peroxidase-conjugated antibodies (1:4,000; #32260; Invitrogen; Thermo Fisher Scientific, Inc.) for 1 h. Protein bands were revealed using a ECL Plus Western Blotting Detection System kit (GE Healthcare Life Sciences, Roosendaal, The Netherlands), with GAPDH used as a loading control. Densitometric analysis of the western blot was performed using Quantity One software, version 4.6.0 (Bio-Rad, Laboratories, Inc., USA), with GAPDH used for normalization.

All cellular experiments were performed at least three times. Data are expressed as the mean ± standard deviation. Statistical analyses were performed using SPSS 14.0 for Windows (SPSS, Inc., Chicago, IL, USA). Experimental and control groups were compared using the unpaired Student's t-test and one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

To assess the inhibitory effect of Fan (Fig. 1A) on the growth and survival of lung cancer cells, human SPC-A-1 lung cancer cells were treated with Fan at concentrations of 1.25, 2.5, 5, 10, 20 and 40 µM for 72 h, using a CCK-8 assay. As shown in Fig. 1B, the proliferative inhibitory effect of Fan was observed in a concentration-dependent manner, with statistical significance (P<0.01 for 5–40 µm). The half-maximal inhibitory concentration value of Fan in SPC-A-1 cells at 72 h was 7.19 µM. Furthermore, phase contrast imaging and Giemsa staining assays were also performed to measure the inhibitory function of Fan treatment (Fig. 2A and B, respectively). Following treatment with 2.5, 5 or 10 µM Fan for 48 h, the total cell number and cell volume of the SPC-A-1 cells decreased in a dose-dependent manner, and morphological changes, such as membrane blebbing, were detected. Thus, Fan appears to inhibit the proliferation of SPC-A-1 lung cancer cells.

To determine whether Fan-induced suppression of cell proliferation was associated with an alteration in cell cycle distribution, the dose-dependent effects of Fan on the cell cycle distribution of lung cancer cells were measured (Fig.3A and B). Following treatment with 2.5, 5 or 10 µM Fan for 48 h, the proportion of SPC-A-1 cells in the G₀/G₁ phase (56.86±0.19, P<0.05; 59.12±1.00, P<0.05; and 66.22±0.32%, P<0.01; respectively) significantly increased, compared with the control (51.84±1.06%); whereas the percentage of cells in the S phase significantly decreased from 15.78±0.17% (control), to 12.42±0.52 (P<0.05), 12.83±0.65 (P<0.05) and 7.96±0.05% (P<0.01), respectively. Furthermore, the proportion of SPC-A-1 cells in the G₂/M phase decreased in a dose-dependent manner from 32.16±0.81% (control) to 30.67±0.70 (P<0.05), 27.52±0.60 (P<0.05) and 25.38±0.29% (P<0.01), respectively. These results indicated that Fan-induced inhibition of SPC-A-1 cell proliferation is cell cycle-dependent, and may result in the enhanced accumulation of cells in the G₀/G1 phase. A representative profile of the cell cycle distribution is outlined in Fig. 3.

D-type cyclins, such as cyclin D1, and its partner kinases CDK4 and CDK6, are central mediators of the G1 phase transition (13). To examine whether the enhancement of G₀/G₁ phase arrest in Fan-treated SPC-A-1 cells was a result of the dysregulation of cell cycle-related genes, the mRNA expression levels of cyclin D1, CDK4 and CDK6 were analyzed. The administration of Fan repressed the expression of cyclin D1, CDK4 and CDK6 mRNAs (Fig. 4A). Fan concentrations of 2.5, 5 and 10 µM significantly inhibited cyclin D1 levels by 19 (P<0.05), 30 (P<0.01) and 57% (P<0.01), respectively, compared with no treatment (Fig. 4B); whereas CDK4 expression levels were inhibited by 9 (P>0.05), 14 (P<0.05) and 27% (P<0.01), respectively (Fig. 4B), and CDK6 expression levels were inhibited by 16 (P>0.05), 19 (P>0.05) and 68% (P<0.01), respectively (Fig. 4B).

The cyclin D1-CDK4/6 complexes formed during the G1 phase may phosphorylate Rb protein and activate a transcriptional factor, E2F-1 (14). Therefore, to determine whether Fan suppressed the expression of cyclin D1, CDK4 and CDK6 via inhibition of the pRB/E2F-1 signaling pathway, the expression levels of pRB and E2F-1 in Fan-treated SPC-A-1 cells were examined, using a western blot assay. As demonstrated in Fig. 4C, treatment with Fan significantly inhibited the expression of pRB protein, and at 2.5, 5 and 10 µM, the suppression rates were 14 (P<0.05), 21 (P<0.05) and 73% (P<0.01), respectively (Fig. 4D). Furthermore, Fan also significantly repressed the expression of E2F-1 protein, and the suppression rates were determined to be 7, 13 (P<0.05) and 61% (P<0.01) at 2.5, 5 and 10 µM, respectively (Fig. 4C and D).