JFK contains 12 herbals described in Table I, and these raw herbs were obtained from the pharmacy dispensary of Longhua Hospital, Shanghai University of Traditional Chinese Medicine (Shanghai, China). The names of herbs have been verified via www.theplantlist.org. Voucher specimens were deposited at the herbarium in Shanghai Jiao Tong University. The raw components of JFK (20 g) were minced and extracted with 200 ml 70% ethanol at 80°C for 1 h according to the composition in Table I. Chromatographic and mass spectrographic finger prints for JFK ethanol extract were examined via LC-MS. The ethanol extract was filtered through a 0.45 µm syringe filter and diluted to various concentrations used in the present study.

The human lung cancer A549, NCI-H1975, NCI-H1650 and NCI-H2228 cell lines were maintained with RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 100 U/ml penicillin and 100 µg/ml streptomycin (Thermo Fisher Scientific, Inc.) at 37°C in a humidified atmosphere containing 5% CO₂.

Human lung cancer cell lines were treated with various concentrations of JFK extract (0, 0.041, 0.054, 0.081, 0.108, 0.162 and 0.216 mg/ml) for 48 h at 37°C, and then the cell viability was evaluated using Cell Counting Kit 8 (CCK8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) by spectrophotometric plate reader (Omega Bio-Tek, Inc., Norcross, GA, USA). All cell viability results were tested by three independent experiments.

To evaluate the effects of JFK extract on cell growth, 1×10⁵ A549 cells were seeded in each well of 6-well plates, and exposed to JFK extract at a half maximal inhibitory concentration (IC50; determined by CCK8 assay) for 48 h. The morphological changes in A549 cells were observed with a phase-contrast microscope (Model Ti-E; NIS4.0; Nikon, Tokyo, Japan). A549 cells were fixed, stained with DAPI and observed using fluorescence microscopy (Model Ti-E; NIS4.0; Nikon).

Briefly, A549 cells were harvested by trypsin with no EDTA (Thermo Fisher Scientific, Inc.), and washed twice with PBS. The cells were fixed with cold 70% ethanol overnight, and then stained with a solution consisting of 20 µg/ml propidium iodine (PI) (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and 0.1% TritonX-100 (Sigma-Aldrich; Merck KGaA), 100 µg/ml RNase A (Fermentas; Thermo Fisher Scientific, Inc.) for 15 min at 37°C in the dark. Cell cycle progression was then analyzed by flow cytometry (BD LSRFortessa; BD Biosciences, Franklin Lakes, NJ, USA) and ModFit LT software (version 3.2; Verity Software House, Topsham, ME, USA).

Determination of phosphatidyl serine (PS) and membrane integrity was performed using the Annexin V-FITC/PI Apoptosis kit (Beijing Zoman Biotechnology Co., Ltd., Beijing, China). In brief, A549, NCI-H1975, NCI-H1650 and NCI-H2228 cells were harvested by trypsin (no EDTA) and washed twice with PBS, then stained with Annexin V-FITC/PI and analyzed by flow cytometry as aforementioned. PI positive cells were designated as end stage apoptotic cells, and FITC-positive cells were designated as early stage apoptotic cells.

RNA-seq assays were performed in A549 cells without and with JFK treatment for 48 h (0.054 mg/ml), as described in our recent study (17). Briefly, total RNA was extracted by TRIzol (Thermo Fisher Scientific, Inc.), and further treated with DNase to remove genomic DNA contamination. Isolation of mRNA was performed with Oligotex mRNA Mini kit (Qiagen, Hilden, Germany) and then used for RNA-seq library construction with NEBNext Ultra Directional RNA Library Prep kit for Illumina (New England BioLabs, Inc., Ipswich, MA, USA), which was subjected to Illumina sequencing. The raw sequencing data are available in the EMBL database (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-3592.

The raw sequencing reads were mapped to the reference genome (hg19) by TopHat. Cufflinks was used to characterize the differential transcription pattern (18). The present study has taken biases in library preparation into account. The gene expression level was measured by reads per kilobase of transcript per million reads mapped.

The Database for Annotation, Visualization and Integrated Discovery (DAVID) bioinformatics resource was used to annotate gene functions and pathways (19,20).

Transcription level of genes of interest was evaluated using quantitative reverse transcription-quantitative polymerase chain reaction (RT-qPCR) with SYBR-Green PCR Master Mix (Qiagen). RT-qPCR was performed according to our previous studies (13,16,17). All primer sequences used for RT-qPCR are presented in Table II.

Cells were washed with PBS and collected in a 1.5 ml tube. A549 cells were lysed using the procedures essentially the same as described previously (13). The total protein extracts were obtained following centrifugation (18,000 × g for 15 min, at 4°C). The protein concentration was evaluated by Pierce BCA protein assay kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Protein samples (~50 µg) were fractionated by SDS-PAGE (10% polyacrylamide gels) and transferred to a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). The protein-binding membrane was incubated in PBS containing 0.1% Tween-20 (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 60 min at 4°C. 5% skim milk was used to block non-specific binding for 3 h at 4°C. This was followed by incubation overnight at 4°C with primary antibody. The primary antibodies used in the present study included; cyclin B1 (CCNB1; cat no. D160234-0100; dilution, 1:250; Sangon Biotech Co., Ltd., Shanghai, China), cyclin-dependent kinase 4 (CDK4; cat no. 11026-1-AP; dilution, 1:100; Proteintech Group, Inc., Wuhan, China), death receptor 4 (DR4; cat no. 24063-1-AP; dilution, 1:100; Proteintech Group, Inc.), Fas (cat no. D220092-0100; dilution, 1:550; Sangon Biotech Co., Ltd.), and GAPDH (cat no. 5174S; dilution, 1:2,000; Cell Signaling Technology, Inc., Danvers, MA, USA). The membrane with protein antibody complexes were washed with PBS containing 0.1% Tween-20, incubated for 2 h with a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (cat no. 14708S; dilution, 1:15,000; Cell Signaling Technology, Inc.) at 4°C. Immunodetection was performed using ECL reagent (Santa Cruz Biotechnology Inc., Dallas, TX, USA) and images were captured and analyzed using the LI-COR Odyssey Infrared Imaging system (LI-COR Biosciences, Lincoln, NE, USA). The protein amounts were semi-quantified by analyzing blot intensity using GAPDH as loading control.

siRNA duplexes were designed to downregulate DR4 and Fas. The DR4-targeting siRNA sequence was 5′-CCUUCAAGUUUGUCGUCGUdTdT-3′ and the Fas-targeting siRNA sequence was 5′-GUGCAGAUGUAAACCAAACdTdT-3′. siRNAs for in vitro transfection were obtained from Shanghai Gene Pharma Co., Ltd. (Shanghai, China). Transfection was performed using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's instructions. Briefly, A549 and NCI-H2228 cells were transfected with 50 nmol/l of siRNAs directed against Fas and DR4 respectively, and incubated for 6 h at 37°C.

Data are presented as the mean ± standard deviation. Comparisons between the groups were examined by standard one-way analysis of variance ANOVA, SPSS for Windows 14.0 software package (SPSS, Inc., Chicago, IL, USA), followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To examine the effects of JFK on lung cancer cell growth, A549, NCI-H1975, NCI-H1650 and NCI-H2228 cells were treated with JFK at various concentrations (0, 0.041, 0.054, 0.081, 0.108, 0.162 and 0.216 mg/ml) for 48 h and cell viability was assessed. The cell viability of these human lung cancer cell lines decreases significantly in a concentration-dependent manner following JFK treatment (Fig. 1A). Additionally, JFK was observed to exert an increased inhibition effect on A549 cells compared with other three human lung cancer cell lines, with a IC50 of ~0.054 mg/ml. JFK treatment (0.054 mg/ml) for 48 h reshapes a large number of A549 cells to be distinctly flat and enlarged in size (Fig. 1B) and makes the nucleus condensed and disintegrated (Fig. 1C), indicating multiple effects of JFK on A549 cells. Based on these observations, A549 was next focused on and the antitumor mechanisms of JFK were characterized.

To further investigate whether JFK cytotoxicity involves cell cycle arrest, a flow cytometry assay was performed to characterize alterations of the cell cycle. As shown in Fig. 1D, cell cycle analysis indicates that a significant portion of A549 cells accumulates in G1 and G2 phase upon JFK (0.054 mg/ml) treatment, suggesting that JFK could induce G1/S and G2/M transition arrest. To examine whether JFK triggers apoptosis in A549 cells, the extent of apoptotic cells was investigated by flow cytometry, and it was observed that numerous A549 cells were induced to undergo apoptosis after exposure to JFK for 48 h (Fig. 1E). This phenomenon not only presents in early apoptosis, but also presents in total apoptosis (Fig. 1F). Thus, the cell apoptotic rates dramatically increase upon JFK treatment.

Given the observations of anti-proliferation and pro-apoptosis effects induced by JFK, the present study subsequently aimed to understand the underlying molecular mechanisms by transcriptome-wide differential gene expression analysis through RNA-seq. In total, 13,130,096 and 10,433,359 reads were generated from JFK-treated and untreated A549 cells, 54 and 66% of which were uniquely mapped to the human genome, corresponding to 13,720 and 17,519 expressed genes, respectively (Table III). Compared with the control, 5,281 differentially expressed genes (P<0.05) were identified, with 2,777 (52.6%) downregulated and 2,504 (47.4%) upregulated (Fig. 2A).

Gene Ontology (GO) analysis of the genes with JFK-induced differential expression was performed using DAVID (19) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways database (20). The top ten GO items were listed in Table IV, in which apoptosis-associated and cell cycle-associated biological processes are statistically enriched in the upregulated and downregulated genes, respectively. For the gene sets with differential expression, KEGG pathway analysis was performed. Similarly, it was observed that the cell cycle and apoptosis pathways are also included in the top 10 enriched pathways (Fig. 2B and C). These results suggest that JFK could induce growth inhibition potentially through repressing pathways of cell cycle, nuclear division and translation pathways. Simultaneously, it could induce apoptosis in A549 cells partially through activating apoptosis, cell death and protein transport pathways.

For gene sets with a differential expression pattern determined by RNA-seq analysis, cell cycle-associated genes, including CDK2, CDK4, CCNB1 and cyclin A2 (CCNA2), and apoptosis-associated genes, including Fas, DR4, tumor protein P53 binding protein 2 (TP53BP2) and BCL2 interacting protein 3 like (BNIP3L), for further validation (Fig. 3A). The transcriptional activities of selected genes were measured by RT-qPCR, and the results were shown in Fig. 3B and C. These results confirm that JFK represses cell cycle-associated genes, and activates apoptosis-associated genes as well. Among these selected genes, the alterations in protein level of CDK4, CCNB1, Fas and DR4 were examined by western blot analysis. As shown in Fig. 3D and E, protein levels of CDK4 and CCNB1 decrease and those of Fas and DR4 increase after JFK (0.054 mg/ml) treatment.

To explore whether Fas and DR4 mediate the JFK-induced apoptosis in A549 cells, lastly siRNA assays were performed to suppress the expression of Fas and DR4 in JFK-treated A549 cells. As shown in Fig. 4A and B, the apoptotic rates (including early apoptosis rate and total apoptosis rate) significantly decrease when Fas and DR4 were knocked down. The protein expression levels of Fas and DR4 also drop when Fas and DR4 were knocked down (Fig. 4C). These results indicate that activation of Fas and DR4 is partially involved in JFK-induced apoptosis.