Cordycepin, also termed 3′-deoxyadenosine, is a nucleoside analogue from
Cordycepin, also termed 3′-deoxyadenosine, is a nucleoside analogue from
Apoptosis, also termed programmed cell death, is a key regulator of tissue homeostasis and is characterized by typical morphological and biochemical hallmarks, including cell shrinkage, membrane blebbing, chromatin condensation and nuclear fragmentation (
Although cordycepin has been shown to have a cytotoxic effect on HepG2 cells, the detailed molecular mechanisms have not been well elucidated (
Dried
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
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
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 (
Cordycepin exhibited the ability to inhibit the proliferation of HepG2 cells in a time- and dose-dependent manner (
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 (
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 (
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 (
As a natural compound, cordycepin plays a key role in cancer therapy and induces apoptosis in numerous cells by targeting molecules and pathways (
It is well known that mitochondria play a major role in several stress-induced cell death pathways, and damage to mitochondria with subsequent loss of mitochondrial membrane potential has been known to be the point of no return in apoptotic cascades (
In mammalian cells, apoptosis occurs via either the extrinsic (receptor-dependent) or the intrinsic (mitochondria-dependent) pathway, with each involving caspase activation. Fas is a TNF-receptor that transduces the apoptotic signal into cells (
Fas may induce caspase-mediated cleavage of p22 Bid into a major p15, and minor p13 and p11 product; the major proteolytic product p15 tBid allows the release of cytochrome
The Bcl-2 family proteins play an essential role in apoptotic progress, such as Bcl-2 protein (anti-apoptosis) and Bax protein (pro-apoptosis). These proteins are regulators of mitochondrial membrane permeability and intermembrane space protein efflux, according to the opposing fractions of the anti-apoptosis members and pro-apoptosis members (
Cleaved Bid causes cytochrome
It has been shown that cordycepin is an analogue of adenosine (
In conclusion, cordycepin-induced apoptosis in HepG2 cells may be initiated by the FADD mediated signal pathway and regulated by the Bcl-2 family proteins that can change the alternation of mitochondrial membrane permeability and cause the mitochondria mediated apoptosis signal pathway (
HPLC and MS analysis of cordycepin. (A) HPLC assay. The retention time of fraction A was 10.5 min, which corresponds with the retention time of cordycepin. (B) MS assay. The m/z of fraction A was 252.1096 Da, which is consistent with the m/z of standard cordycepin. HPLC, high performance liquid chromatography; MS, mass spectrometric.
Antiproliferative effect of cordycepin on HepG2 cells. The cell viability of HepG2 cells were measured subsequent to treatment with the indicated concentrations of cordycepin for 24, 48, 72 and 96 h. Cordycepin produced growth inhibitory effects on HepG2 cells. *P<0.05, **P<0.01 vs. 24 h cordycepin treatment.
Analysis of the effect of cordycepin on HepG2 cells by DAPI and PI staining. (A) Effect of cordycepin on HepG2 cells, as determined by DAPI staining. HepG2 cells were fixed and stained with DAPI and the nuclear morphology of the cells was then examined with a fluorescence microscope subsequent to treatment for 48 h with the indicated concentrations of cordycepin. Cytoplasmic shrinkage and nuclear condensation were observed in the cordycepin group, due to the increased fluorescent intensity compared with the control group. (B) Effect of cordycepin on the sub-G1 proportion of HepG2 cells, as determined by PI staining. HepG2 cells were fixed and stained with PI for flow cytometric analysis subsequent to treatment for 48 h with the indicated concentrations of cordycepin. The percentage of cells with a hypodiploid DNA content (sub-G1) represent those fractions undergoing apoptotic DNA fragmentation. DAPI, 4′,6-diamidino-2-phenylindole; PI, propidium iodide.
Mitochondrial membrane potential, as assessed by JC-1 staining. HepG2 cells were fixed and stained with JC-1 staining for a flow cytometric analysis subsequent to treatment for 48 h at the indicated concentrations of cordycepin.
Western blot analysis of the signaling pathway involved in cordycepin-induced apoptosis. (A) Effects of cordycepin on the expression levels of Fas, FasL, FADD and caspase-8. (B) Effects of cordycepin on the expression level of the Bcl-2 family proteins. (C) Effects of cordycepin on the expression level of cyto C, caspase-9 and caspase-3. FasL, Fas ligand; FADD, Fas-associated death domain protein; Bcl-2, B-cell lymphoma-2; Bax, Bcl-2-associated X protein; Bid, BH3 interacting domain death agonist; t-Bid, truncated Bid; cyto C, cytochrome
Overview of the pathways involved in cordycepin-induced apoptosis in HepG2 cells. FADD, Fas-associated death domain protein; Bcl-2, B-cell lymphoma-2; Bax, Bcl-2-associated X protein; Bid, BH3 interacting domain death agonist; t-Bid, truncated Bid; cyto C, cytochrome