Hirsutella sinensis extract (0.199 g/ml), which was produced by fermentation and extraction using ethanol, water and enzyme digestion methods by Huadong Medicine (Hangzhou, Zhejiang, China), was provided by Dr Shenglang Zhu at Nanshan Hospital (Shenzhen, Guangdong, China). Hirsutella sinensis is the anamorphic, mycelial form of Cordyceps sinensis (25,26). Cordycepin (catalog no., C3394) and 3-methyladenine (catalog no., M9281) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

The human neuroblastoma cell line SK-N-SH was obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Word Federation for Culture Collection registered no., 793; Shanghai, China). The human neuroblastoma cell line BE(2)-M17 was from the Cell Resource Center (Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China). The cells were cultured using standard methods (27) in Dulbecco's modified Eagle's medium (DMEM; Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA), 2 mmol/l L-glutamine (Sigma-Aldrich), 100 units/ml penicillin and 100 µg/ml streptomycin (Gibco), at 37°C in a 5% CO₂ atmosphere.

The pEGFP-LC3 plasmid (plasmid no., 21073; Addgene, Inc., Cambridge, MA, USA) is a pEGFP-C1 plasmid (Clontech Laboratories, Inc., Mountainview, CA, USA) inserted with a 738-bp long cDNA sequence from rat microtubule-associated protein 1 light chain 3 (LC3). This sequence encodes a fusion protein composed of green fluorescent protein (GFP) at the N-terminus and LC3 at the C-terminus (28). In total, 1×10⁶ SK-N-SH cells/well were seeded into six-well plates, 24 h prior to transfection. The SK-N-SH cells were transfected with pEGFP-LC3 using FuGENE HD Transfection Reagent (Promega Corporation, Madison, WI, USA) and left for 48 h, followed by selection with 800 µg/ml G418 (Amresco, Inc., Framingham, MA, USA) for one week.

The SK-N-SH or BE(2)-M17 cells were seeded at a density of 1×10⁶ cells/well into six-well plates 24 h prior to treatment. SK-N-SH cells were treated with various concentrations of Cordyceps sinensis extract. The SK-N-SH and BE(2)-M17 cells were treated with various concentrations of cordycepin. The morphology of each cell line was analyzed using an Axiovert 40 microscope (Zeiss, Oberkochen, Germany).

The SK-N-SH or BE(2)-M17 cells were seeded at a density of 10⁴ cells/well in 96-well plates, 24 h prior to treatment. At 48 h subsequent to cordycepin treatment, the medium was removed and 100 µl of serum free DMEM containing 0.5 mg/ml MTT (Sigma-Aldrich) was added to each well. The cells were then incubated at 37°C for 4 h. Subsequent to the removal of the MTT media, 100 µl dimethyl sulfoxide was added to each well and incubated at 37°C for 20 min. The plate was incubated at room temperature for an additional 5 min with agitation, and the absorbance of the solution was subsequently quantified at a wavelength of 570 nm, which was normalized against a reference value measured at a wavelength of 690 nm to account for debris, using the PARADIGM Detection Platform (Beckman Coulter, Brea, CA, USA).

The SK-N-SH cells were cultured in DMEM supplemented with 10% FBS, 2 mmol/l L-glutamine, 100 units/ml penicillin and 100 µg/ml streptomycin at 37°C in a 5% CO₂ atmosphere. The SK-N-SH cells (5×10⁵) were seeded into each well of a six-well plate and grown overnight, which was followed by treatment with 0, 50, 100 or 200 µmol/l cordycepin for 48 h. For the experiment measuring the effect of 3-methyladenine on cordycepin-induced apoptosis, SK-N-SH cells were treated with 0 or 150 µmol/l cordycepin, 5 mmol/l 3-methyladenine, or combination of the latter two, for 48 h. Untreated cells were used as the negative control. The free-floating and attached SK-N-SH cells from each well were harvested and pooled. The cells were washed with phosphate buffered saline (PBS) and stained using a Dead Cell Apoptosis kit with Annexin V Alexa Fluor 488 and propidium iodide (PI) (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer's instructions. The stained cells were analyzed using a Cytomics FC 500 flow cytometer (Beckman Coulter).

The antibodies against caspase-3 (catalog no., 9665S), poly(adenosine diphosphate-ribose) polymerase 1 (PARP1; catalog no., 9542S) and LC3 A/B (catalog no., 4108S) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The anti-β-actin antibody (catalog no., sc-47778) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), and the whole-molecule anti-rabbit immunoglobulin (Ig)G-peroxidase (cat. no. A9169) and whole-molecule anti-mouse IgG-peroxidase antibodies (cat. no. A9044) were purchased from Sigma-Aldrich.

In total, 1×10⁶ cells were lysed in 150 µl cell lysis buffer (Cell Signaling Technology) containing an EDTA-free protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland). The protein concentrations were determined using a detergent-compatible protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). For each well, 30 µg of protein lysate was combined with 2 µl 2X sample buffer, comprising final concentrations of 60 mmol/l Tris (pH, 6.8), 10% glycerol, 2% sodium dodecyl sulfate and 5% mercaptoethanol. The lysate was then incubated at 100°C for 5 min to denature the proteins. The protein samples were loaded for electrophoresis, and then transferred to Immobilon-P membranes (Millipore, Billerica, MA, USA) using wet transfer apparatus. The membrane was blocked in 1% bovine serum albumin (Sigma-Aldrich) in PBS for 2 h at room temperature. The membranes were subsequently probed with primary antibodies overnight at 4°C, followed by secondary antibody incubation at room temperature for 1.5 h. The protein bands were visualized with enhanced chemiluminescence prime western blotting detection reagent (Amersham, GE Healthcare, Shanghai, China) and images were captured using the MiniChemi professional machine (SageCreation Science, Co., Ltd., Beijing, China).

Student's t-test was used to compare the viability of SK-N-SH and BE(2)-M17 cells measured by MTT assay. Statistical analyses were performed using Microsoft Excel 2010 software (Microsoft, Redmond, WA, USA). The results are presented as the mean ± standard error of the mean. All P-values were two-tailed and P<0.05 was considered to indicate a statistically significant difference.

To examine the effects of Hirsutella sinensis extract on neuroblastoma cells, various concentrations of the extract, comprising 0, 0.001, 0.01 and 0.1 mg/ml, were incubated with SK-N-SH cells for 48 h. Administration of 0.1 mg/ml Hirsutella sinensis extract markedly altered the cell phenotype, as indicated by an increased proportion of SK-N-SH cells possessing cell granulations that were visible under a phase-contrast microscope, compared with the untreated control (Fig. 1). Administration of 0.001 and 0.01 mg/ml Hirsutella sinensis extract resulted in no significant effect on the phenotype of the SK-N-SH cells (Fig. 1).

The effect of the purified cordycepin chemical, one of the bioactive components of Cordyceps sinensis, on the neuroblastoma cells was also investigated. The SK-N-SH cells were treated with 0, 50, 100 and 200 µmol/l cordycepin for 48 h, and the cell viability was assessed using an MTT assay. The viability of the SK-N-SH and BE(2)-M17 cells was significantly reduced as the cordycepin concentration increased (Fig. 2A). Examination of the SK-N-SH and BE(2)-M17 cells by phase contrast microscopy also revealed an increased number of floating cells and reduced the number of attached cells following treatment with cordycepin, compared with that of non-treated cells (Fig. 2B).

In order to investigate whether apoptosis occurs in cordycepin-treated neuroblastoma cells, SK-N-SH cells were incubated with or without cordycepin and analyzed by FACS following staining with Annexin V-PI. The percentage of early-apoptotic cells in the SK-N-SH cell populations treated with 100 or 200 µmol/l cordycepin was significantly increased (29.8%), compared with the percentage of early-apoptotic cells (10.3%) in the negative control (Fig. 3A). As BE(2)-M17 cells tend to form clusters and do not separate well, apoptosis in this cell line was not assessed by FACS.

The expression of the apoptosis markers caspase-3 and PARP1 was examined by western blot analysis (29). As shown in Fig. 3B, the expression of the 35 kDa full length caspase-3 protein and its cleavage products were upregulated in the SK-N-SH cells treated with 50, 100 and 200 µmol/l cordycepin, compared with the expression in the non-treated control cells. Similar results were obtained from the BE(2)-M17 cells treated with 50 or 100 µmol/l cordycepin. However, in the BE(2)-M17 cells treated with 200 µmol/l cordycepin, caspase-3 upregulation and cleavage was decreased.

In the SK-N-SH and BE(2)-M17 cells, expression of full-length PARP1 was not altered by cordycepin at concentrations up to 200 µmol/l. A significant increase in cleavage of PARP1 was observed in SK-N-SH cells treated with 50, 100 or 200 µmol/l cordycepin for 48 h, compared with untreated cells (P<0.001)(Fig. 3B). In the BE(2)-M17 cells treated with 50, 100 or 200 µmol/l cordycepin, PARP1 cleavage increased in a dose-dependent manner, compared with the control samples (Fig. 3B).

The effect of cordycepin treatment on autophagy in the neuroblastoma SK-N-SH cells was assessed using fluorescence microscopy. As shown in Fig. 4A, the SK-N-SH cells that were stably transfected with pEGFP-LC3 and treated with 200 µmol/l cordycepin for 72 h exhibited a marked shift from a diffuse to a punctate pattern of LC3-associated green fluorescence (Fig. 4A), indicating that autophagy occurs in cordycepin-treated SK-N-SH cells.

Western blot analysis also revealed increased LC3 A/B protein cleavage in the SK-N-SH cells treated with 200 µmol/l cordycepin compared with the untreated controls (Fig. 4B). However, treatment with 40 µmol/l of cordycepin exhibited no significant effect on autophagy in SK-N-SH cells (Fig. 4B).

3-methyladenine was previously reported to be an autophagy inducer under nutrient-rich conditions (13), which may negatively affect apoptosis. Thus, we investigated the effect of 3-methyladenine on cordycepin-induced apoptosis. The results of FACS revealed that the percentage of late-apoptotic cells in SK-N-SH cell populations treated with 150 µmol/l cordycepin and 5 mM 3-methyladenine was significantly reduced (12.8%), compared with the percentage of late-apoptotic cells (18.1%) in SK-N-SH cells treated with 150 µmol/l cordycepin alone (Fig. 5).