Dried Cordyceps militaris was purchased from Cordyceps Garden Biotechnology Company (Fuzhou, China). The macroprous adsorption resin NKA-II was purchased from the Chemical Plant of Nankai University (Tianjin, China). Sulforhodamine B (SRB), 4′,6-diamidino-2-phenylindole (DAPI), propidium iodide (PI) and tetraethylbenzimidazolylcarbocyanine iodide (JC-1) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The following antibodies were purchased from Sangon Biotech Co., Ltd. (Shanghai, China): Mouse monoclonal anti-Fas (1:3,000; catalog no. D198888); rabbit polyclonal anti-Fas ligand (1:3,000; catalog no. D262701); mouse monoclonal anti-Fas associated with death domain protein (FADD; 1:3,000; catalog no. D199671); rabbit polyclonal anti-caspase-8 (1:2,000; catalog no. D155240); rabbit polyclonal anti-caspase-9 (1:2,000; catalog no. D220078); rabbit ployclonal anti-caspase-10 (1:2,000; catalog no. D260010); rabbit monoclonal anti-caspase-3 (1:3,000; catalog no. D120074); mouse monoclonal anti-B-cell lymphoma-2 (Bcl-2; 1:3,000; catalog no. D198628); rabbit polyclonal anti-Bcl-2 associated X protein (Bax; 1:3,000; catalog no. D120073); mouse monoclonal anti-BH3 interacting domain death agonist (Bid; 1:3,000; catalog no. D198911); and rabbit polyclonal anti-cytochrome c (1:2,000; catalog no. D110006). Rabbit polyclonal anti-mouse IgG (1:3,000; catalog no. sc-358920) and goat anti-rabbit IgG (1:3,000; catalog no. sc-2768) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The Mitochondrial Membrane Potential Assay kit with JC-1, radioimmunoprecipitation assay (RIPA) lysis buffer and Cell Mitochondria Isolation kit were purchased from Beyotime Institute of Biotechnology (Haimen, China).

HepG2 cell lines were purchased from the Culture Center of the Institute of Basic Medical Sciences of the Chinese Academy of Medical Sciences (Beijing, China). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.), 100 IU/ml penicillin and 100 µg/ml streptomycin (Sigma-Aldrich), and cultured at 37°C for 48 h in a humidified atmosphere consisting of 95% air and 5% CO₂.

A total of 150 g dried fruiting bodies of C. militaris were cleaned with distilled water, dried in a dark and ventilated place and crushed prior to soaking in 500 ml distilled water overnight at room temperature, and then boiled in distilled water for 3 h. Subsequent to filtration to remove debris, the filtrate (crude aqueous extract) was concentrated in a rotary evaporator (model no. 0010001820; IKA^® Works Guangzhou, Guangzhou, China). Ethanol was added and the mixture was left at room temperature overnight. The precipitate obtained subsequent to filtration was discarded and the supernatant was lyophilized to yield the crude sample. The crude extract was chromatographed on a NKA-II column (Sigma-Aldrich) and eluted with 50% (v/v) ethanol to collect the fractions, which were then further purified by high performance liquid chromatography (HPLC). The Lab Alliance HPLC system (model no. 2690/2695; Waters Technologies Ltd., Shanghai, China), including two Series III pumps (model no. 90–2489 rev M; Scientific Systems, Inc. State College, PA, USA) and anabsorbance detector (model no. 500; Waters Technologies Ltd.) set at 254 nm, connected to a Cs420 Hardware integrator (model no. Cs420; Waters Technologies Ltd.). The column was a 250×10 mm reversed-phase Merck Millipore C18 ODS column (Merck Millipore, Darmstadt, Germany) with an internal diameter of 12 µm. The temperature was set at 30°C and the elution conditions were as follows: Flow rate, 0.8 ml/min; and solvent, 15% methanol.

Various concentrations of cordycepin (0, 125, 250 and 500 µM) were inoculated into 96-well microplates when the cell concentration was adjusted to 1×10⁵ cells/ml. Cultures in triplicate were treated with cordycepin for 48 h and the control wells received only maintenance medium (DMEM). Cellular responses were colorimetrically evaluated by an SRB assay. Briefly, the cells were fixed with 25% trichloroacetic acid (Sigma-Aldrich) and washed and stained with 0.4% SRB. Subsequent to the cell-bound SRB being solubilized by the addition of 10 mM Tris-HCl, bound SRB was colorimetrically assessed using an ELISA microplate reader (MK3; Thermo Fisher Scientific, Inc.) at 490 nm. Cell growth inhibition was expressed as a percentage of the untreated control absorbance following the subtraction of the mean background absorbance. Compounds were considered to have potent growth inhibitory activity when the reduction in SRB absorbance was >25% compared with the untreated control cells. The half maximal inhibitory concentration (IC₅₀) values were calculated from the dose-response curves.

HepG2 cells were seeded into a 50 ml culture flask at a density of 1×10⁵ cells/ml for 24 h. Subsequent to treatment with or without cordycepin (0, 125, 250 and 500 µM) at 37°C for 48 h, the cells were collected and washed with phosphate-buffered saline (PBS). Cell pellets were fixed with 4% paraformaldehyde for 10 min and washed three times with PBS. The cells were then incubated with 5 µg/ml DAPI for 20 min. Subsequent to washing with PBS, the cells were observed under a fluorescence microscope (model no. Bx51; Olympus Corporation, Tokyo, Japan) and images were captured.

HepG2 cells cultured with or without cordycepin at 37°C for 48 h were harvested, washed with PBS and fixed with 70% cold ethanol at 4°C for 4 h. The fixed cells were washed and stained with a PI solution containing 20 µg/ml PI and 10 mg/ml RNase (Takara Biotech, Inc., Dalian, China) in PBS for 20 min in the dark. The stained cells were then analyzed by flow cytometry using fluorescence-activated cell sorting (FACS) and the FACSCalibur system (BD Biosciences, Franklin Lakes, NJ, USA). In addition, subsequent to being treated with various concentrations of cordycepin (0, 125, 250 and 500 µM) for 48 h, the harvested HepG2 cells were washed in PBS and incubated in a freshly prepared JC-1 solution at 37°C for 20 min. Cell-associated fluorescence was also measured by FACS.

HepG2 cells were treated for 48 h with various concentrations of cordycepin (0, 125, 250 and 500 µM) and harvested and lysed in RIPA lysis buffer that contained 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS. The cell lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to a polyvinylidene fluoride membrane, and then blocked for 1 h with 5% fat-free milk in PBS with Tween-20 (PBST) at room temperature. The membrane was immunoblotted overnight with antibodies in PBST at 4°C. Subsequent to being washed with PBST, the membrane was detected by enhanced chemiluminescence (Kodak, Rochester, NY, USA). Equal loading was confirmed by probing with an antibody against β-actin (1:3000; catalog no. sc-8432; Santa Cruz Biotechnology, Inc.). Mitochondrial and cytosolic fractions were prepared in order to detect cytochrome c. Following treatment with the indicated concentrations of cordycepin for 48 h, HepG2 cells were harvested and disposed using the Cell Mitochondria Isolation kit, according to the manufacturer's protocol. Finally, the mitochondrial pellet and cytosolic supernatant were separated using a centrifuge (model no. 5415C; Eppendorf, Hamburg, Germany).

Two fractions from crude extract, termed A and B, were eluted using NKA macroporous resin (data not shown). Fraction A was then purified on a HPLC preparative column followed by an analytical column using CH₃OH:H₂O = 15:85 (v/v) as the mobile phase. The retention time of fraction A was ~10.5 min, which is consistent with that of standard cordycepin (Fig. 1A). In addition, mass spectrometric analysis revealed that the m/z of fraction A was 252.1096 Da, which corresponds with the m/z of standard cordycepin (Fig. 1B).

Cordycepin exhibited the ability to inhibit the proliferation of HepG2 cells in a time- and dose-dependent manner (Fig. 2). The IC₅₀ values for HepG2 cells treated with cordycepin for 24, 48, 72 and 96 h were 735±3.67, 497.5±2.49, 385±1.93 and 307.5±1.54 µM, respectively.

The fluorescence microscopic examination with DAPI staining revealed that morphological changes, such as chromatin condensation, nuclear shrinkage or fragmentation and apoptotic body formation, occurred in HepG2 cells subsequent to being treated with cordycepin for 48 h (Fig. 3A).

The flow cytometric analysis with PI staining revealed that the cells treated with various concentrations of cordycepin for 48 h exhibited an apoptotic hypodiploid sub-G1 peak in a concentration-dependent manner (Fig. 3B). The flow cytometric analysis with JC-1 staining showed the respective green fluorescence intensity ratios, which indicated the apoptotic HepG2 cells, were 5.72, 18.58, 32.12 and 36.65%, respectively (Fig. 4).

Cordycepin increased the expression levels of Fas and FADD, but had no effect on FasL expression. Additionally, the level of procaspase-8 decreased, while the level of cleaved caspase-8 was elevated (Fig. 5A). The expression of the Bcl-2 family proteins was also detected and the results showed that cordycepin enhanced the expression level of truncated Bid (tBid) and decreased the level of Bid, but had little effect on the levels of Bax and Bcl-2 (Fig. 5B). A decreased level of cytochrome c was detected in the mitochondrial fraction and an increased level was detected in the cytosolic fraction (Fig. 5C). Furthermore, the results also showed that HepG2 cells treated with cordycepin for 48 h underwent significant cleavage of precaspase-9 and precaspase-3 (Fig. 5C).