ONCOLOGY LETTERS 12: 995-1000, 2016 Cordycepin induces apoptosis in human liver cancer HepG2 cells through extrinsic and intrinsic signaling pathways LE-WEN SHAO1, LI-HUA HUANG1, SHENG YAN2, JIAN-DI JIN3 and SHAO-YAN REN3 1Nursing Department; Departments of 2Hepato-Biliary-Pancreatic Surgery and 3Infectious Disease, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, P.R. China Received March 6, 2015; Accepted April 12, 2016 DOI: 10.3892/ol.2016.4706 Abstract. Cordycepin, also termed 3'-deoxyadenosine, is a to have natural medicinal properties, such as anti-angiogenic, nucleoside analogue from Cordyceps sinensis and has been anti‑tumor, anti‑viral, anti‑inflammatory and hypoglycemic reported to demonstrate numerous biological and pharma- effects (1-8). cological properties. Our previous study illustrated that the Cordycepin, also termed 3'-deoxyadenosine, is a nucleo- anti-tumor effect of cordycepin may be associated with apop- side analogue from C. militaris (9) and has been reported tosis. In the present study, the apoptotic effect of cordycepin to demonstrate numerous notable biological and pharma- on HepG2 cells was investigated using 4',6-diamidino-2-phe- cological properties, including immunological stimulation, nylindole, tetraethylbenzimidazolylcarbocyanine iodide and anti-cancer and anti-viral effects (10-15), a stimulating effect propidium iodide staining analysis and flow cytometry. The on interlukin-10 production as an immune modulator (16) and results showed that cordycepin exhibited the ability to inhibit preventing hyperlipidermia (17). HepG2 cells in a time- and dose-dependent manner when cells Apoptosis, also termed programmed cell death, is a key produced typical apoptotic morphological changes, including regulator of tissue homeostasis and is characterized by chromatin condensation, the accumulation of sub-G1 cells and typical morphological and biochemical hallmarks, including change mitochondrial permeability. A potential mechanism for cell shrinkage, membrane blebbing, chromatin condensation cordycepin-induced apoptosis of human liver cancer HepG2 and nuclear fragmentation (18). The mechanisms for foreign cells may occur through the extrinsic signaling pathway medi- chemicals disrupting cell functions and causing tissue damage ated by the transmembrane Fas-associated with death domain have been associated with the triggering of apoptosis signal protein. Apoptosis was also associated with Bcl-2 family transduction pathways, consisting of the intrinsic (mitochon- protein regulation, leading to altered mitochondrial membrane drial) and extrinsic (death receptor) pathways (19). permeability and resulting in the release of cytochrome c Although cordycepin has been shown to have a cytotoxic into the cytosol. The activation of the caspase cascade is effect on HepG2 cells, the detailed molecular mechanisms responsible for the execution of apoptosis. In conclusion, have not been well elucidated (17,19). In the present study, cordycepin-induced apoptosis in HepG2 cells involved the the mechanistic understanding of how cordycepin mediates extrinsic and intrinsic signaling pathway and was primarily apoptosis in HepG2 cells was investigated and the intrinsic regulated by the Bcl-2 family proteins. and extrinsic apoptosis pathways were the main focus. Introduction Materials and methods Cordyceps militaris is a fungus that parasitizes Lepidoptera Materials. Dried Cordyceps militaris was purchased from larvae and has been extensively used as a folk tonic food and Cordyceps Garden Biotechnology Company (Fuzhou, China). crude drug (1). Cordyceps militaris has long been considered 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 tetraethylbenzimidazolyl- carbocyanine iodide (JC-1) were purchased from Sigma-Aldrich Correspondence to: Dr Li-Hua Huang, Nursing Department, The First Affiliated Hospital, College of Medicine, Zhejiang University, (St. Louis, MO, USA). The following antibodies were purchased 79 Qingchun Road, Hangzhou, Zhejiang 310003, P.R. China from Sangon Biotech Co., Ltd. (Shanghai, China): Mouse E-mail: [email protected] monoclonal anti-Fas (1:3,000; catalog no. D198888); rabbit polyclonal anti-Fas ligand (1:3,000; catalog no. D262701); Key words: Cordyceps sinensis, cordycepin, antitumor, apoptosis, mouse monoclonal anti-Fas associated with death domain HepG2 protein (FADD; 1:3,000; catalog no. D199671); rabbit poly- clonal 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. 996 SHAO et al: CORDYCEPIN INDUCES APOPTOSIS IN HepG2 CELLS D260010); rabbit monoclonal anti-caspase-3 (1:3,000; catalog were fixed with 25% trichloroacetic acid (Sigma-Aldrich) no. D120074); mouse monoclonal anti-B-cell lymphoma-2 and washed and stained with 0.4% SRB. Subsequent to the (Bcl-2; 1:3,000; catalog no. D198628); rabbit polyclonal anti- cell-bound SRB being solubilized by the addition of 10 mM Bcl-2 associated X protein (Bax; 1:3,000; catalog no. D120073); Tris-HCl, bound SRB was colorimetrically assessed using an mouse monoclonal anti-BH3 interacting domain death agonist ELISA microplate reader (MK3; Thermo Fisher Scientific, Inc.) (Bid; 1:3,000; catalog no. D198911); and rabbit polyclonal at 490 nm. Cell growth inhibition was expressed as a percentage anti-cytochrome c (1:2,000; catalog no. D110006). Rabbit of the untreated control absorbance following the subtraction of polyclonal anti-mouse IgG (1:3,000; catalog no. sc-358920) and the mean background absorbance. Compounds were considered goat anti-rabbit IgG (1:3,000; catalog no. sc-2768) antibodies to have potent growth inhibitory activity when the reduction were purchased from Santa Cruz Biotechnology, Inc. (Dallas, in SRB absorbance was >25% compared with the untreated TX, USA). The Mitochondrial Membrane Potential Assay kit control cells. The half maximal inhibitory concentration (IC50) with JC-1, radioimmunoprecipitation assay (RIPA) lysis buffer values were calculated from the dose-response curves. and Cell Mitochondria Isolation kit were purchased from Beyo- time Institute of Biotechnology (Haimen, China). Assessment of apoptosis by DAPI staining. HepG2 cells were seeded into a 50 ml culture flask at a density of 1x105 cells/ml Cell lines and culture. HepG2 cell lines were purchased from for 24 h. Subsequent to treatment with or without cordycepin the Culture Center of the Institute of Basic Medical Sciences (0, 125, 250 and 500 µM) at 37˚C for 48 h, the cells were of the Chinese Academy of Medical Sciences (Beijing, China). collected and washed with phosphate-buffered saline (PBS). The cells were maintained in Dulbecco's modified Eagle's Cell pellets were fixed with 4% paraformaldehyde for 10 min medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., and washed three times with PBS. The cells were then incu- Waltham, MA, USA) supplemented with 10% fetal bovine bated with 5 µg/ml DAPI for 20 min. Subsequent to washing serum (Invitrogen; Thermo Fisher Scientific, Inc.), 100 IU/ml with PBS, the cells were observed under a fluorescence micro- penicillin and 100 µg/ml streptomycin (Sigma-Aldrich), and scope (model no. Bx51; Olympus Corporation, Tokyo, Japan) cultured at 37˚C for 48 h in a humidified atmosphere consisting and images were captured. of 95% air and 5% CO2. Assessment of apoptosis by flow cytometry. HepG2 cells Isolation and purification of cordycepin. A total of 150 g cultured with or without cordycepin at 37˚C for 48 h were dried fruiting bodies of C. militaris were cleaned with distilled harvested, washed with PBS and fixed with 70% cold ethanol water, dried in a dark and ventilated place and crushed prior to at 4˚C for 4 h. The fixed cells were washed and stained with soaking in 500 ml distilled water overnight at room tempera- a PI solution containing 20 µg/ml PI and 10 mg/ml RNase ture, and then boiled in distilled water for 3 h. Subsequent to (Takara Biotech, Inc., Dalian, China) in PBS for 20 min in filtration to remove debris, the filtrate (crude aqueous extract) the dark. The stained cells were then analyzed by flow cytom- was concentrated in a rotary evaporator (model no. 0010001820; etry using fluorescence‑activated cell sorting (FACS) and the IKA® Works Guangzhou, Guangzhou, China). Ethanol was FACSCalibur system (BD Biosciences, Franklin Lakes, NJ, added and the mixture was left at room temperature overnight. USA). In addition, subsequent to being treated with various The precipitate obtained subsequent to filtration was discarded concentrations of cordycepin (0, 125, 250 and 500 µM) for and the supernatant was lyophilized to yield the crude sample. 48 h, the harvested HepG2 cells were washed in PBS and incu- The crude extract was chromatographed on a NKA-II column bated in a freshly prepared JC‑1 solution at 37˚C for 20 min. (Sigma-Aldrich) and eluted with 50% (v/v) ethanol to collect Cell‑associated fluorescence was also measured by FACS. the fractions, which were then further purified by high perfor- mance liquid chromatography (HPLC). The Lab Alliance Western blot analysis. HepG2 cells were treated for 48 h HPLC system (model no. 2690/2695; Waters Technologies with various concentrations of cordycepin (0, 125, 250 and Ltd., Shanghai, China), including two Series III pumps (model 500 µM) and harvested and lysed in RIPA