Cordycepin was purchased from Chengdu Pufei De Biotech Co., Ltd. (China). Fetal bovine serums was purchased from Hangzhou Sijiqing Biological Engineering Materials Co. (China). Dulbecco's modified Eagle's medium (DMEM) were purchased from (Gibco, China), 3–4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were from Sigma Chemical Co. (St. Louis, MO, USA). Propidium iodide (PI), Annexin V-FITC Apoptosis Detection kit, rhodamine 123 and reactive oxygen species kit were from Beyotime Institute of Biotechnology (Shanghai, China). Rabbit polyclonal anti-human Bcl-2, anti-human Bax, P-PI3K, Akt, P-Akt, caspase-8, antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse, anti-rabbit, caspase-3 p-JAK2 (catalog no. bs-2485R), p-Stat3 (catalog no. bs-1658R) were purchased from Bioss (Beijing Biosynthesis Biotechnology), p53, MDM2, survivin, Cdk2, Cdk1, and cyclin E antibodies were purchased from Santa Cruz Biotechnology, DR3, A₃AR antibodies were purchased from Kanghexin Biotech Co. (Suzhou, China).

We followed the ethical classification standards and our laboratory guidelines; SGC-7901 (gastric cancer) cell line was purchased from ATCC (Shanghai, China).

SGC-7901 cells were seeded and cultured in 10-cm dish with DMEM medium contained 10% FBS (Gibco), then incubated in an incubator (humidified 37°C, the atmosphere of 5% CO₂). Cells were allowed to grow to 70–80% before used.

SGC-7901 cells were seed and cultured in a 96-well plate to a final concentration of 5×10³ with DMEM medium with 1% FBS then incubated at 37°C for 18–24 h. After all, cells were treated with the appropriated concentration of cordycepin for 24 h. Afterward, the cell medium was discarded, and fresh medium was added to each well, with 20 µl MTT solutions (5 mg/ml), and then incubated at 37°C for 4 h. Finally, 150 µl of DMSO was added to each well, incubated in the dark for 10 min, then read at the wavelength of 570 nm using Varioskan Flash Multimode Reader (Thermo Scientific, USA). The absorbance was easured at 570 nm, and results were expressed as a percentage, relative to solvent-control incubations, and the IC₅₀ were determined using non-linear regression analysis (percentage survival versus concentration).

For Annexin V/PI assays, SCG-7901 cells were stained with Annexin V-FITC and PI, and evaluated for apoptosis by flow cytometry according to the manufacturer's protocol (Beyotime, China). After treatment with 0, 20, 40 and 80 µM of cordycepin, and incubation at 37°C for 24 h, SGC-7901 cells were collect and washed twice with PBS, then stained with 5 µl of Annexin V-FITC and 10 µl of PI in 500 µl binding buffer for 15 min at room temperature in the dark. The apoptotic cells were determined using flow cytometry, and the data were analysed using Cellquest analysis software.

SGC-7901 cells were cultured in 12-well plates. After 24 h, cells were treated with cordycepin for 24 h, and then fixed in 4% cold paraformaldehyde (PFA) for 30 min. Subsequently, SGC-7901 cells were incubated with high DAPI (1 mg/ml) for 30 min in the dark; then, the cells were washed with PBS. Apoptotic nuclei characterized as intensively stained were detected using fluorescent microscopy (model IX71; Olympus, Tokyo, Japan).

The cell cycle distribution in different phases after exposure of cordycepin were analysed by flow cytometry. In brief, SGC-7901 cells were harvested and washed with PBS after exposure to 0, 20, 40 and 80 µM of cordycepin for 24 h. Subsequently, the cells were fixed with 70% of ethanol at −20°C for 2 h and then stained with PI solution consisting of 1 mg/ml PI and RNase A. The fluorescence-activated cells were analysed by the flow cytometry, and the data were analyzed using Cellquest analysis software.

The ROS generation was measured after staining the cells with DCFH-DA and flow cytometry. Briefly, after exposure of different concentrations of cordycepin, SGC-7901 stained by 1 ml of PBS containing 10 µM DCFH-DA, and incubated for 30 min at 37°C. The fluorescence emission from DCF was analysed via Cellquest analysis software flow cytometry (Cytomics FC 500; Beckman Coulter Inc., Miami, FL, USA), with excitation and emission spectra set at 480 and 530 nm, respectively.

The MMP was measured using a flow cytometry (Cytomics FC 500; Beckman Coulter Inc.), and the fluorescent dye rhodamine 123. In brief SGC-7901 cells were cultured in 12-well plates, after treatment with cordycepin cells were harvested, and washed with PBS. Then stained with Rh123 (100 µg/l) for 30 min at 37°C. The cells were collected by pipetting and washed twice with PBS and analysed by flow cytometry.

SGC-7901 cells were treated with 0, 20, 40 and 80 µM cordycepin in DMEM medium with 1% FBS incubated for 24 h. The cells were harvested and collected on ice-cold PBS; further RIPA containing proteinase inhibitor cocktail was added in cells, incubated on ice for 30 min. Afterward, insoluble protein lysate was removed by centrifugation at 1,350 rpm for 15 min at 4°C. The protein concentrations were measured by using NanoDrop 1000 (Thermo Scientific) spectrophotometer, 70 µg of proteins were resolved on 10–12% SDS-PAGE and transferred to PVDF membranes. After blocking with 5% (w/v) non-fat milk and washing with a Tris-buffered saline-Tween solution (TBST), membranes were incubated with respective primary antibodies at 4°C overnight and washed three times with TBST. The blots were then incubated with anti-rabbit or anti-mouse horseradish peroxidase conjugated secondary antibodies for 1 h at room temperature. Finally membrane was washed again with TBST three times; signals were detected using ECL plus chemiluminescence kit on X-ray film (Millipore Corp., Billerica, USA) (28).

Statistical analysis was performed using Origin lab 8. Each experiment was repeated at least three times. All data are presented as the mean ± standard deviation (SD). Statistical significance was evaluated using one-way analysis of variance (ANOVA). Differences were considered to be statistically significant at P<0.05.

MTT assay was used to assess the cell viability of SGC-7901 cells in the presence of several concentrations of cordycepin (Fig. 1A) ranging from 0 to 100 µM. The growth inhibition improved consistently with time over the period of the incubation time. Specifically, the estimated half-maximal inhibitory concentration (IC₅₀) values were 40, 32 and 7 µM after 24, 48 and 72 h, correspondingly (Fig. 1B). The cytotoxicity of cordycepin was further observed after the SGC-7901 cells were treated with 0, 20, 40 and 80 µM cordycepin. As seen in Fig. 1C cordycepin inhibited SGC-7901 cell proliferation and cells death rate increased with incensement of cordycepin concentration.

Loss of membrane plasma and DNA fragmentation are the key cha racteristics possessed by apoptotic cell death. The impact of cordycepin on SGC-7901 cells death was evaluated by DNA fragmentation with the help of DAPI staining and fluorescent microscopy. As seen in Fig. 2A cordycepin incorporates the shape of the SGC-7901 cells modified to a considerable extent via increasing dose-dependently. Particularly, cordycepin broke the cell membranes leading to inducing the nuclear condensation by apoptotic in comparison with the control cells. Induction of apoptosis was further validated by Annexin V/PI assay. This is based on the probe of the initial apoptosis (B4), late apoptosis (B2), and necrosis (B1) of SGC-7901 cells. The results indicated that the B4 values increased 8.091±1.435, 24.37±1.829, 49.33±1,492 and 89.74±2.714% by utilizing 0, 20, 40 and 80 µM of cordycepin, correspondingly (Fig. 2B).

Additionally, to evaluate whether cordycepin-induced apoptosis was dependent upon mitochondrial pathways, western blot analysis was carried out to monitor protein expression of mitochondrial extrinsic apoptosis. As shown in Fig. 2C, treatment of SGC-7901 cells by cordycepin, induces activation of death receptor DR3. Indeed, in the absence of cordycepin DR3 protein expression is null while increased together with the cordycepin concentration. This activation of DR3 encouraging activation of caspase-8, that functions as an initiator of caspase-3, further enhancing cleavage of PARP consequently inducing SGC-7901 cell death by apoptosis (14).

The phosphatidylinositol 3-kinase/Akt pathways are engaged in the SGC-7901 tumor expansion and its metastasis. Especially, the inhibition of Akt results in disturbance of the biological activities of SGC-7901 cells by mediating their cell cycle arrest (29). A previous report, supported that the activation of DR3 in colon cancer cells, leads to activation of PI3K inducing cell apoptosis (30). We therefore, evaluated whether the activation of DR3 in SGC-7901 cells was associated with activation of PI3K/Akt signaling. As presented in Fig. 3, cordycepin inhibits the expression level of PI3K, considerably decreasing P-Akt protein expression. At the same time, cordycepin boosts the expression of p38 whereas Akt remains almost the same. These findings show that cordycepin is likely to induce SGC-7901 death by mediating their cells cycle arrest, and this through inhibition of AKT and increased p38.

PKM2 is a key enzyme that regulates aerobic glycolysis in tumor cells and particularly in gastric cancer. Its inhibition was report to affect SGC-7901 cells growth (31). We therefore, investigated the expression of PKM2 in SGC-7901 cells. As shown in Fig. 5, cordycepin suppressed PKM2 expression. We can therefore suggest that this inhibition of PKM2 by cordycepin contributed to the induction apoptosis in SGC-7901 cells.

A₃AR is a normal purine metabolite extensively expressed in most cancer cells. Its inhibition is involved in inhibiting the proliferation and induction of cell death by apoptosis (32). To gauge whether the apoptosis mediated by cordycepin possesses the capacity to influence A₃AR expression in SGC-7901 cells western blot analysis was carried out and results showed that cordycepin inhibited drastically the protein expression of A₃AR expression (Fig. 2C). Based on this we suggest that inhibition of A₃AR is likely to play a role in the induction of apoptosis mediated by cordycepin on SGC-7901 cells.

The Jak-Stat cascade proteins are essential for inflammatory as well as immune responses of anticancer agents (33). The impacts of cordycepin on the Jak-Stat protein expression was probed with the help of western blot analysis. The findings brought to light that cordycepin improved the phosphorylation of Jak2 and Stat3 proteins in SGC-7901 cells (Fig. 3). This is clearly due to the potential of cordycepin to translocate Stat3 and Jak2 from the cytoplasm into the nucleus leading to the initiation of the gene expression of pro-inflammatory response.

To validate whether mitochondrial events were involved in induction of apoptosis, flow cytometry of rhodamine 123 staining and western blot analysis was carried out. As depicted in Fig. 4A, during the unavailability of NAC, the mitochondrial membrane potential expression is minimized to a significant extent. On the other hand, the mitochondrial membrane potential expression was restored in the availability of NAC upon utilization of cordycepin (0, 20, 40 and 80 µM) for 24 h, recommending that mitochondrial were taking part in cordycepin induced SGC-7901 cell apoptosis. To confirm our findings immunoflorescence of Rhodamine were further processed. As seen in Fig. 4B, cordycepin induce SC-7901 cells morphology changes due to the lost of mitochondrial membrane metabolite resulting in their collapse.

Moreover, to develop more understanding of the process by which cordycepin decreased mitochondrial membrane potential, western blot analysis is carried out for the purpose of validating the level of cytochrome c, Bax, and Bcl-2 to obtain more insight into cell apoptosis. The findings suggest that cordycepin resulted in release of cytochrome c from the mitochondrial membrane which is the major factor in the development of apoptosomes, which trigger the activation of Bax and deactivates Bcl-2 (Fig. 4C).

Previous study on cordycepin shows that ROS is linked to a collapse of mitochondrial membrane potential (34). We carried out an analysis of the production of intracellular ROS level with the help of flow cytometry to evaluate whether apoptosis was caused by cordycepin. Specifically, the cells treated with cordycepin were loaded with the fluorescent probes DCF-DA to gauge the H₂O₂ in the availability as well as unavailability of NAC and incubated for 24 h. The findings show that, in the absence of NAC, the ROS values are 18.22±1.52, 43.14±1.52, 61.39±1.73 and 83.23±2.075 upon 0, 20, 40 and 80 µM of cordycepin, correspondingly. On the other hand, in the presence of NAC, the generation of ROS expression are 45.72±2.08 and 49.57±1.25 via utilization of 40, and 80 µM cordycepin correspondingly denoting the role of NAC in minimizing H₂O₂ (Fig. 5). These results validate the role of cordycepin in the generation of oxidative stress in SGC-7901 cells.

To validate whether the inhibition of Akt can influence SGC-7901 cell cycle arrest, flow cytometry was carried out. In the four cell cycle phases, the S phase, M phase, G1, and G2 phase, the treatment of SGC-7901 cell line with 0, 20, 40, and 80 µM for 24 h induce an increase in the S phase percentages to 45.29±2.13, 53.315±2.54, 71.092±2.24 and 71.203±3.1. These concentrations also resulted in the decrease of the G0/G1 and G2/M phase percentages. Based on the above evidence, we confirmed that cordycepin induces SGC-7901 cell cycle arrest at the S phase (Fig. 6A). To further delineate and validate the process of the SGC-7901 cell cycle arrest by cordycepin, the protein expression of cyclin E, CDK1 and CDK2, cyclin D1, p53, p21 MDM2, and pRb proteins were explored by western blot analysis. Fig. 6B suggests that cordycepin suppressed significantly the expression of cyclin E, CDK1, CDK2, MDM2, cyclin D1, as well as phosphorylated pRb. At the same time, cordycepin augmented the p53 and p21 expression. These findings indicated that cordycepin induces SGC-7901 cell progression at S phase (Fig. 6A).