Cordycepin and cisplatin were purchased from MedChemExpress. Immediately before use, cordycepin was dissolved in media to generate a 10 mM stock solution, and cisplatin was dissolved in N,N-dimethylformamide to generate a 10 mM stock solution. Both stock solutions were stored at −20°C. The Human C666-1 NPC cell line, which is an EBV-positive NPC cell line taken from undifferentiated NPC tissue, was obtained from the cell line database (Shanghai FuHeng Biological Technology Co., Ltd.), and were cultured in RPMI-1640 media (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc.), penicillin (100 U/ml) and streptomycin (100 U/ml) (both Gibco; Thermo Fisher Scientific, Inc.) in a humidified incubator at 37°C (5% CO₂).

C666-1 cells were seeded into a 96-well plate (5×10³ cells per well) and treated with increasing concentrations of cordycepin (0, 250, 500, 750 and 1,000 µM for 24–72 h, or cisplatin for 24 h at 37°C. Following treatment, cell viability was assessed using the Cell Counting Kit 8 (CCK-8) assay (APExBIO Technology LLC); 10 µl CCK-8 solution was added, and the cells were incubated for 4 h at 37°C. Optical density was detected at 450 nm using a microplate reader (Thermo Fisher Scientific, Inc.).

C666-1 cells were counted, seeded into 12-well plates in triplicate (800 cells per well), cultured in RPMI (supplemented with 10% fetal bovine serum), and treated with cordycepin for up to 14 days as aforementioned. Then, the cells were washed twice with PBS and fixed using methanol for 10 min at 4°C. After two additional washes with PBS, the cells were stained with crystal violet for 30 min at room temperature. The cells were then washed with double-distilled water (ddH₂O) to remove the crystal violet, and the colony numbers were counted using ImageJ software (version 1.52; National Institutes of Health), set to the area of colonies above 5 pixel^{^2} as 1 colony.

C666-1 cells were seeded into 12-well plates and incubated in serum-free medium at 37°C for 18 h. The cell monolayers were scratched with a 10-µl pipette tip and washed with serum-free RPMI-1640 media (Gibco; Thermo Fisher Scientific, Inc.) to remove cells detached from the plates. The cells were incubated in the presence or absence of cordycepin for 48–72 h (as aforementioned) in medium containing 10% FBS. Then, the medium was replaced with PBS and images of the cells were captured using a fluorescence inverted microscope (Leica Microsystems, Inc.; magnification, ×20) in brightfield mode. The results were quantified using ImageJ software (version 1.52).

To assess cellular migration, 5×10⁴ cells were seeded into Transwell inserts in a 24-well plate, with serum-free medium in the upper chambers, and RPMI containing 10% FBS added to the lower chambers. The cells were incubated for 24–48 h at 37°C, washed once with PBS, and then fixed with 4% paraformaldehyde for 10 min at room temperature. The cells were stained with 0.1% crystal violet for 30 min at room temperature, and then washed with ddH₂O. The non-migrated cells were removed with a cotton swab, and the stained cells were observed by a fluorescence inverted microscope (Leica Microsystems, Inc.; magnification, ×20).

Total RNA of each cordycepin-treated and control sample was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), following the manufacturer's instructions. The quality of RNA was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.) and checked using RNase-free agarose gel electrophoresis. Then mRNA was enriched and purified using oligo (dT) beads. The purified mRNA was cut into short fragments using fragmentation buffer (Shanghai Yeasen Biotechnology Co., Ltd.) and reverse transcribed into cDNA using random primers. Second-strand cDNA were synthesized using DNA polymerase I, RNase H, dNTP and buffer. Then the cDNA fragments were purified using the QIAquick PCR extracting kit (Qiagen). After performing end repair and adding poly (A), the cDNA fragments were ligated using Illumina sequencing adapters. The ligation products were enriched via PCR amplification to construct the cDNA library template (NovaSeq 6000 S4 Reagent Kit v1.5; 300 cycles; cat. no. 20028312). Finally, the library (10 pM per sample) was sequenced by 150 bp paired end sequencing using the Illumina Novaseq 6000 (Illumina Inc.) by Guangzhou Gene Denovo Biotechnology Co. Ltd.

Sequencing reads were edited for quality and cleaned using fastp (version 0.18.0, http://github.com/OpenGene/fastp). Clean data were mapped to the Homo sapiens (human) genome (GRCh38.p13) using HISAT2 (version 2.4, http://daehwankimlab.github.io/hisat2/). The mapped reads of each sample were assembled using StringTie (version 1.3.1, http://ccb.jhu.edu/software/stringtie) using a reference-based approach. For each transcription region, a FPKM (fragment per kilobase of transcript per million mapped reads) value was calculated to quantify its expression abundance and variation using RSEM software (http://deweylab.biostat.wisc.edu/rsem). Differentially expressed genes were identified using DESeq2 (http://www.bioconductor.org/) and edgeR (http://www.rproject.org/) package, with a threshold false discovery rate <0.05, and absolute value of the log2 fold change ≥1. All expressed genes were functionally annotated against the NCBI non-redundant protein database using the BLAST algorithm with a cut-off E-value ≤10⁻⁵. The genes were also subjected to classification and enrichment analyses of Gene Ontology (GO) functions and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. GO and KEGG classification were performed using Gene Ontology database (http://geneontology.org/) and the KEGG automatic annotation server (https://www.genome.jp/kegg/), respectively.

Cells were harvested and lysed in RIPA buffer (Sangon Biotech Co., Ltd.) supplemented with protease inhibitor (1% phenylmethylsulfonyl fluoride) at 4°C, for 30 min, and then centrifuged at 10,309 × g for 15 min 4°C. The protein concentrations of the lysates were measured on a spectrophotometer using a BCA Protein Assay Kit (Sangon Biotech Co., Ltd.). The remainder were added to a 5X loading buffer at 1:4 (Sangon Biotech Co., Ltd.) and heated at 95°C for 5 min. Next, 50 µg protein per lane was electrophoretically separated by 10% SDS-PAGE, and transferred to PVDF membranes. The membranes were blocked using 5% non-fat milk for 1 h at room temperature, and the incubated with primary antibodies against GAPDH (1:2,000; cat. no. AP0063; Bioworld Technology, Inc.) ERK1/2 (1:1,000; cat. no.137F5; Cell Signaling Technologies, Inc.), p-ERK1/2 (1:1,000; cat. no.9101; Cell Signaling Technologies, Inc.) and β-catenin (1:1,000; cat. no. ab16051; Abcam) in 1X TBS with 0.05% Tween (TBS-T), at 4°C overnight. The membranes were then incubated with HRP-conjugated secondary antibodies (anti-rabbit; 1:5,000; cat. no. 7074; Cell Signaling Technologies, Inc.) in 1X TBS-T at room temperature for 1 h. The proteins were visualized using a chemiluminescence (ECL) reagent (Pierce ECL Western Blotting Substrate; Thermo Fisher Scientific, Inc.).

RNA sequencing (RNA-seq) results were validated using RT-qPCR. Total RNA was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) per the manufacturer's instructions. Reverse transcription and qPCR were performed using an RT Kit (Hunan Accurate Bio-Medical Co., Ltd.) and TB Green PCR Master Mix (Takara Bio, Inc.), respectively, according to the manufacturers' protocols. The qPCR reaction was carried out using a three-step method on a LightCycler 480 Instrument II (Roche Diagnostics): Initial denaturation at 95°C for 5 min, then amplification at 95°C for 5 sec, 58°C for 30 sec and 72°C for 20 sec (a total of 40 cycles). The oligonucleotide primers are displayed in Table I; GAPDH was used as the housekeeping gene to normalize the expression levels of mRNA, which were quantified using the 2^−ΔΔCq method (18).

Each experiment was repeated three times independently, and the data are expressed as the mean ± standard deviation. The data were analyzed using GraphPad Prism 6.02 (GraphPad Software, Inc.); differences between two groups were analyzed using the unpaired Student's t-test, and differences among ≥3 groups were compared using one-way ANOVA followed by Dunnett's multiple comparisons test. P<0.05 was considered to indicate a statistically significant difference.

C666-1 cells, EBV-positive NPC cells taken from undifferentiated NPC tissue (19), were used to investigate the effects of cordycepin on NPC cell proliferation. Increasing concentrations of cordycepin (250, 500 and 1,000 µM) were added to the culture media for 24, 48 and 72 h, and C666-1 cell viability was determined using a CCK-8 assay. The results demonstrated that cordycepin decreased the viability of C666-1 cells in a dose-dependent manner, with an IC50 of 1.37 mM at 24 h, 842.5 µM at 48 h and 546.9 µM at 72 h (Fig. 1A). Furthermore, a colony formation assay revealed that 500 µM cordycepin significantly inhibited colony formation, and that 1 mM cordycepin completely inhibited clone formation (Fig. 1B and C). These results indicate that cordycepin inhibited the proliferation of NPC cells.

To investigate the effects of cordycepin on the migration of C666-1 cells, wound-healing and Transwell assays were performed. Cells treated with 250 and 500 µM cordycepin showed inhibited wound-healing ability (Fig. 2A and B). Furthermore, 500 µM cordycepin significantly inhibited C666-1 cell migration through the Transwell insert membrane (Fig. 2C). These results indicate that cordycepin inhibited the migration of NPC cells.

Next, the potential for cordycepin to enhance the chemotherapeutic effects of cisplatin was investigated in NPC cells. An additional colony formation assay was performed, and a relatively low concentration of cisplatin that did not affect cell viability (0.5 µg/ml) was used. The assay revealed that the colony formation ability of NPC cells was completely inhibited following combined treatment with cordycepin and cisplatin (Fig. 3). This result indicates that cordycepin enhanced the inhibitory effects of cisplatin on NPC cells.

To investigate how cordycepin regulates downstream signaling pathways, transcriptome RNA-seq analyses were performed to compare cells treated with 500 µM cordycepin for 48 h with untreated control cells (Fig. 4A). In total, 20,295 genes were identified, and the expression levels of 1,541 genes were altered to varying degrees. Among the differentially expressed genes, 72 were significantly different (Log2|FoldChange |> 2), P<0.05), including 35 downregulated and 37 upregulated genes (Fig. 4B). The top upregulated and downregulated genes are presented in Fig. 4C. This experiment showed global gene expression alterations following cordycepin treatment.

To map changes in the downstream signaling pathways, GO and KEGG pathway enrichment analyses were performed on all significantly differentially expressed genes (above a 1.5-fold change). An overview of the top functions is provided in Fig. 5. Both up- and downregulated genes were enriched, revealing a global map of signaling transduction changes in the cells.

A detailed enrichment figure reveals KEGG pathways that could affect the proliferation and migration of cancer cells, including ‘cell adhesion molecules’, ‘PD-L1 expression and PD-1 checkpoint pathway in cancer’, ‘T cell receptor signaling pathway’, ‘TNF signaling pathway’, and ‘VEGF signaling pathway’. GO term enrichment results highlighted biological processes related to cell proliferation and migration, such as “regulation of the JNK cascade’, ‘positive regulation of the stress-activated MAPK cascade’ and ‘cell adhesion’ (Fig. 6A).

Next, Gene Set Enrichment Analysis of the KEGG pathways was performed to identify those that were the most inhibited by cordycepin treatment. These included ‘ribosome’, ‘oxidative phosphorylation’, ‘protein export’, ‘PPAR signaling pathway’, ‘glutathione metabolism’ and ‘central carbon metabolism in cancer’ (Fig. 6B).

These analyses indicate downstream signaling pathway alterations that are regulated by cordycepin.

To verify the RNA-seq data, four significantly differentially expressed genes identified from RNA-seq were selected and verified via RT-qPCR. Selectin E and actin like 10 were upregulated by 500 µM cordycepin treatment, while regulator of G protein signaling 9 and EF-hand domain family member D1 were downregulated, which was consistent with the sequencing results (Fig. 7A). To investigate whether cordycepin affects the MAPK/ERK and Wnt signaling pathways, western blotting was performed to evaluate the protein expression levels of ERK1/2 and phosphorylated-ERK1/2 (p-ERK) at different concentrations of cordycepin for 48 h. The results showed a similar expression level of p-ERK at 500 µM cordycepin compared with the control group, while significant inhibition of ERK and p-ERK1/2 was found after treatment with 750 µM cordycepin, which is close to the IC50 value at this time point (Fig. 7B). The protein expression of β-catenin was also arrested (relative to the untreated control group) following cordycepin treatment. Different expression levels of β-catenin were detected at low (250 µM) and high (500 and 750 µM) concentrations, which suggests different regulation mechanisms of signaling transduction. These results indicate that cordycepin may inhibit proliferation and migration of NPC cells through the ERK and β-catenin signaling pathways.