HCT116 human colorectal carcinoma cells were purchased from the American Type Culture Collection (Manassas, VA, USA). p53^−/−, Bax^−/− and p21^−/− HCT116 cells were obtained from Dr Bert Vogelstein (Johns Hopkins University, Baltimore, MD, USA). The pEGFP-C3-Bax expression vectors were provided by Dr Quan Cheng (Institute of Zoology, Chinese Academy of Sciences, Beijing, China).

Wild-type (WT) HCT116, HCT116-p53^−/−, HCT116-p21^−/− and HCT116-Bax^−/− cells were cultured in McCoy's 5A Medium (cat. no. A1324-9050; AppliChem, Inc., Maryland Heights, MO, USA) with 10% (v/v) fetal bovine serum (GE Healthcare Life Sciences, Logan, UT, USA) and 100 U penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in a 5% CO₂ incubator.

Cordycepin (C₁₀H₁₃N₅O₃; 251 Da; cat. no. C3394; Fig. 1A) and caspase-3 inhibitor (cat. no. 219007) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Rabbit monoclonal antibodies against Bax (cat. no. 5023), pro-caspase-3 (cat. no. 9665), cytochrome c oxidase IV (CoxIV; cat. no. 4850) and cleaved poly(ADP-ribose) polymerase (PARP; cat. no. 9541) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The anti-mouse monoclonal p53 antibody (cat. no. sc-126) and anti-mouse cytochrome c (cat. no. sc-126) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The anti-mouse monoclonal β-actin antibody (cat. no. AC004) was purchased from ABclonal Biotech Co., Ltd. (Woburn, MA, USA). The anti-mouse monoclonal β-tubulin antibody (cat. no. AbM59005-37B-PU) was obtained from Beijing Protein Innovation (Beijing, China). The horseradish peroxidase (HRP)-conjugated secondary antibodies (cat. nos. 111-035-003 or 115-035-003) were obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA).

Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was used for transient transfections according to the manufacturer's protocols. Transfection reagents and DNA were mixed in Opti-MEM (Invitrogen; Thermo Fisher Scientific, Inc.); For 6-well plate, 200 µl (10 µg/ml) transfection DNA complex was added to cells grown to 30–60% confluency, and the culture was incubated for ~3 h, and the medium was replaced with fresh full McCoy's 5A medium. For EGFP-Bax stable transfections, 0.5 mg/ml G418 was added to the medium for 48 h following transient transfection, and the cells were selected after 2 weeks. Thereafter, stable cells were always maintained in 0.25 mg/ml G418 medium.

Soft agar and colony formation assays were used to examine the viability and tumorigenicity of HCT116 cells following treatment with cordycepin. Briefly, 3×10³ HCT116 cells were treated with various concentrations of cordycepin (0, 62.5, 135, 270 and 540 µM) for 24 h; the medium and drugs were subsequently replaced with fresh medium. After 2 weeks incubation, cell clones were stained with 0.05% crystal violet at room temperature for 30 min and images were captured by scanner (MRS-2400U2; Microtek, Shanghai, China).

For the soft agar test, 2 ml of 0.7% lower agar-McCoy's 5A with cordycepin (0–540 µM, as aforementioned) was plated onto each well of 6-well plates. Subsequently, 1 ml of HCT116 cells (1×10⁴) was mixed with 1 ml of 0.7% agar-McCoy's 5A/cordycepin (0–540 µM) mix and added to the curdled lower agar; 2 ml of McCoy's 5A medium was added to the upper agar, and the plates were incubated at 37°C in a 5% CO₂ incubator for 3 weeks, and the numbers of clones were counted and images captured by an inverted microscope (ICX41; SDPTOP, Shanghai, China).

Cell viability was examined using Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan), following the manufacturer's protocol. The HCT116 cell lines [wild-type (WT) HCT116, HCT116-p53^−/−, HCT116-p21^−/− and HCT116-Bax^−/−] aforementioned were seeded (1×10⁴ cells/well) into a 96-well plate, cultured to 60% confluency and exposed to different doses of cordycepin (0, 33.75, 62.5, 135, 270 and 540 µM) for 24 h. The medium was replaced with 100 µl of fresh McCoy's 5A full medium with 10% CCK-8 reagent and incubated for 1 h. Absorbance was detected at 450 nm using an ELx800 microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). The results were expressed as percentages of cell viability.

The HCT116 cell lines (1×10⁴ cells/well) were seeded in 6-well plates and cultured to 60% confluency. Following exposure to the various concentration of cordycepin (0, 135 and 270 µM) for 24 h, cells were collected, washed with phosphate buffered saline (PBS) twice. Subsequently, the cells were centrifuged at 120 × g in the room temperature for 5 min and suspended in apoptosis binding buffer (50 mM HEPES, 700 mM NaCl, 12.5 mM CaCl₂, pH 7.4) at a concentration of 1×10⁶ cells/ml. Cells were stained with propidium iodide (PI) and Annexin V fluorescein isothiocyanate (FITC) for 10–15 min at room temperature. Samples were subjected to flow cytometry (Beckman Coulter, Fullerton, CA) and data were analyzed using FlowJo software (10.07 version). For apoptosis inhibition assay, the cells were incubated with 25 µM caspase-3 inhibitor for 24 h to prevent PARP cleavage and cell apoptosis.

HCT116 cells (5×10⁶⁾ were exposed to 270 µM cordycepin for 24 h. The Mitochondria/Cytosol Fractionation Kit (Beyotime Institute of Biotechnology, Beijing, China) was used to isolate the mitochondrial and cytosolic proteins. Briefly, cells were resuspended in the mitochondria isolation buffer and homogenized with a 2 ml glass homogenizer on ice. Cell lysates were centrifuged at 1,000 × g for 10 min at 4°C, and the supernatant was centrifuged at 14,000 × g for 15 min at 4°C. The supernatant included the cytosolic fraction, and the pellet contained the mitochondrial fraction.

GFP-Bax plasmids were stably expressed in Bax^−/− HCT116 cells. Cells were exposed to cordycepin (0 or 270 µM) for 12 h and fixed with 4% paraformaldehyde for 30 min at room temperature. The fixed cells were observed using a confocal microscope using an ×60 oil objective. Images were captured using 2.0b Olympus FluoView software (Olympus Corporation, Tokyo, Japan), and distribution pattern of GFP-Bax was examined.

An immunoblotting assay was performed as described previously (25). Briefly, cells were cultured on 6-well plates to 60% confluency and subsequently treated with cordycepin (0–540 µM) for 24 h. Cells were lysed with SDS sample buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol) at 95°C for 10 min. Protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific, Inc.). Protein samples (20 µg) were separated by 10% SDS-PAGE, transferred and incubated with indicated primary antibodies (1:1,000) overnight at 4°C and HRP-conjugated secondary antibodies (1:10,000) at room temperature for 1 h. The β-actin was used as loading control. Protein expressions were visualized with the Immobilon Western Chemiluminescent HRP Substrate kit (Merck KGaA, Minneapolis, MN, USA).

All data were analyzed using SPSS software version 20.0 (IBM Corp., Armonk, NY, USA). For data with a Gaussian distribution, parametric statistical analysis was performed using the two-tailed Student's t-test for two groups; one-way analysis of variance was used for multiple comparisons followed by Bonferroni's post hoc analysis for data meeting homogeneity of variance or with Tamhane's T2 analysis for data of heteroscedasticity. For data sets with skewed distribution, non-parametric statistical analysis was performed using the Kruskal-Wallis test followed by the Dunn's test for multiple comparisons. Only in the cases in which the global null hypothesis was rejected was pairwise comparison subsequently performed. For all analyzed variables, a pre-specification approach was performed. P<0.05 was considered to indicate a statistically significant difference.

To determine the most suitable concentration of cordycepin to treat colon cancer cells, the half-maximal inhibitory concentration (IC₅₀) of cordycepin in HCT116 cells was determined. HCT116 cells were treated with 0–540 µM cordycepin for 24 h and further cultured at 37°C in a 5% CO₂ incubator for ~10 days. The viability of the HCT116 cells clearly decreased following cordycepin treatment (Fig. 1B and C); and the results demonstrated that the IC₅₀ of cordycepin was 434 µM for HCT116 cells (Fig. 1B). Soft agar assays were conducted to examine the effects of cordycepin treatment on tumor formation in vitro. At 2 weeks post-treatment, anchorage-independent tumor growth was significantly inhibited in a dose-dependent manner in cells incubated with cordycepin (Fig. 1D). These results demonstrated that cordycepin has significant inhibitory effects on human HCT116 cell viability, proliferation and tumorigenesis in vitro.

To determine the molecular mechanism underlying the anticancer activity of cordycepin, cordycepin-activated apoptotic activity was examined in HCT116 cells. The results demonstrated that cells treated with 0, 135 and 270 µM cordycepin for 24 h exhibited average 7.8, 13.4 and 22.1% early apoptotic rates, respectively (Fig. 2A). To confirm the apoptotic phenotype, the levels of certain proteins that are closely associated with apoptosis were tested. HCT116 cells were exposed to 0–540 µM cordycepin for 24 h, and the protein expression levels of p53 and its target gene p21 were investigated by western blot analysis, which demonstrated notably higher protein expression levels in cells treated with cordycepin, and the increased expressions were in a dose-dependent manner (Fig. 2B). The apoptosis markers, pro-caspase-3 and cleaved PARP were also analyzed by western blotting, and the results demonstrated lower pro-caspase-3 and higher cleaved PARP levels in response to increased cordycepin concentrations (Fig. 2B). These data suggested that cordycepin induced apoptosis in HCT116 cells. To further demonstrate that increased cleaved-PARP occurred through a caspase-3-dependent pathway, cells were incubated with 25 µM caspase-3 inhibitor for 24 h to prevent cell apoptosis, and the results demonstrated that the caspase-3 inhibitor blocked cordycepin-induced cleaved-PARP expression in HCT116 (Fig. 2C). These results suggested that cordycepin may induce apoptosis in HCT116 CRC cells.

Bax, a pro-apoptosis Bcl-2 family member, translocates from the cytoplasm to the mitochondria in response to stimulation, which subsequently results in the release of cytochrome c and cell apoptosis (26). The tumor suppressor protein p53 serves an important role in the maintenance of genomic stability and cell apoptosis (27,28). To understand whether p53 or Bax serves a role in cordycepin-induced apoptosis, the apoptotic effects following cordycepin treatment in HCT116-WT, HCT116-p53^−/− and HCT116-Bax^−/− cells were examined (Fig. 3). HCT116 cells were treated with 0–270 µM cordycepin for 24 h and cell viability was examined (Fig. 3A). The cell viability of HCT116 and HCT116-p53^−/− cells was significantly decreased with increasing cordycepin concentration; however, Bax^−/− cells treated with various concentrations appeared to be insensitive to cordycepin treatment compared with the HCT116-WT and p53^−/− cells. These results indicated that Bax may serve a more essential part in cordycepin-induced cell death compared with p53. In addition, the mutant cell lines were exposed to 62.5 µM cordycepin for 24 h, cultured for an additional 10 days and the colony number reduced in HCT116-WT and HCT116-p53^−/− cells but not in HCT116-Bax^−/− cells (Fig. 3B). HCT116-WT, p53^−/− and Bax^−/− HCT116 cells were also incubated with 270 µM cordycepin for 24 h, and the Annexin V/FITC staining results demonstrated that average 2.76% of the Bax^−/− cells were early apoptotic compared with 8.03% of the HCT116-WT cells, which also suggested that HCT116-Bax^−/− cells were insensitive to cordycepin treatment (Fig. 3C). The reason why p53^−/− cells are more sensitive to cordycepin treatment is that p53 is an essential gene for cell life activities; thus, the amount of cell death in this line is already substantially higher than that of WT cells. Apoptosis will result in DNA fraction; therefore, we determined the DNA fraction using PI staining and the results consistent with the Annexin V/FITC staining (Fig. 3D). These results indicated that cordycepin-induced apoptosis may be Bax dependent.

As aforementioned, cordycepin-induced apoptosis may be Bax-dependent. To assess whether cordycepin induces the cleavage of PARP in p53^−/− or Bax^−/− HCT116 cells, cleaved-PARP protein expression levels were analyzed. Cells were treated with 0–270 µM cordycepin for 24 h, and the protein expression levels of p53, Bax and cleaved-PARP were notably increased with higher concentrations of cordycepin treatment in HCT116-WT cells. Additionally, significant levels of cell apoptosis were observed in the p53^−/− HCT116 cells, but apoptosis markers cleaved-PARP were blocked in the Bax^−/− HCT116 cells (Fig. 4A). The inhibition of cordycepin-induced PARP protein cleavage in the Bax^−/− HCT116 cells was rescued by reintroducing the Bax gene into Bax^−/− HCT116 cells (Fig. 4B). The translocation of Bax to the mitochondria may exert an important effect on apoptotic initiation (29). The stable EGFP-Bax cell line allowed for the tracking of the location of Bax protein expression. Untreated control cells exhibited a diffuse distribution of green fluorescence among whole cells, whereas almost 70% of the cordycepin-treated cells demonstrated only punctate fluorescence, which suggested the translocation of Bax reached 70% in the cordycepin-treated cells (Fig. 4C). In addition, western blot analysis revealed increased Bax protein expression in the mitochondria when exposed to cordycepin (Fig. 4D). cytochrome c is located in the mitochondrial inner membrane; its translocation from the mitochondria to the cytosol induces cell apoptosis (30). As a downstream marker of Bax aggregation, the protein levels of cytochrome c in the cytoplasm of HCT116 cells exposed to cordycepin was examined. The results demonstrated increased cytochrome c expression the cytosol in response to cordycepin exposure (Fig. 4E). These data demonstrated that Bax is employed in cordycepin-induced apoptosis.

In addition to investigating cordycepin-induced apoptosis, whether cordycepin was involved in cell cycle profile changes was examined. Flow cytometric assay results demonstrated that cordycepin treatment resulted in cell cycle arrest in the HCT116-WT cells in the G₁/S phase (Fig. 4F). However, in the p21^−/− HCT116 cells, G₁/S phase arrest was reduced. Whether p53, which is directly controlled by the expression of p21 (31), is involved in G₁/S phase arrest was also examined. As expected, in p53^−/−deficient HCT116 cells, G₁/S phase arrest was clearly ameliorated when compare to HCT116-WT cells treated with 270 µM cordycepin (Fig. 4F). As the protein expression levels of p21 and p53 were increased in response to cordycepin, it was hypothesized that cordycepin treatment may induce G₁/S phase cell cycle arrest in HCT116 cells by inducing the expression of p53 and its target gene p21.