Cordycepin (MW, 251.2; product no. C3394) from C. militaris, 4′,6-diamidino-2-phenylindole (DAPI), N-acetyl-L-cysteine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 5,5′,6,6′-tetra- chloro-1,1′,3,3′ tetraethylbenzimidazolylcarbocyanine iodide (JC-1) and propidium iodide (PI) were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Caspase activity assay kits were obtained from R&D Systems (Minneapolis, MN, USA). An enhanced chemiluminescence (ECL) kit and RNeasy kit were purchased from Amersham Corp. (Arlington Heights, IL, USA) and Qiagen (La Jolla, CA, USA), respectively. RPMI-1640 medium and fetal bovine serum (FBS) were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA) and Gibco-BRL (Gaithersburg, MD, USA), respectively. All other chemicals were purchased from Sigma-Aldrich. Antibodies against cIAP-1, cIAP-2, XIAP, Bcl-2, Bcl-xL, Bax, Bid, poly(ADP-ribose) polymerase (PARP), β-catenin, capase-3, -8 and -9 were purchased form Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Antibodies against extracellular signal-regulated kinase (ERK), phospho (p)-ERK, p38 mitogen-activated protein kinase (MAPK), p-p38 MAPK, JNK, p-JNK, Akt and p-Akt were purchased from BD Biosciences (San Jose, CA, USA). Antibody against actin was from Sigma-Aldrich. Peroxidase-labeled donkey anti-rabbit and sheep anti-mouse immunoglobulin were purchased from Amersham Corp.

Hep3B human hepatocellular carcinoma cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured at 37ºC in a 5% CO₂ humidified incubator, and maintained in RPMI-1640 culture medium containing 10% heat-inactivated FBS. For the cell viability assay, cells were grown to 70% confluence and treated with the indicated concentrations of cordycepin, TRAIL, or a combined treatment (cordycepin plus TRAIL). After treatment, MTT working solution was added to 6-well culture plates and incubated continuously at 37ºC for 2 h. The culture supernatant was removed from the wells, and DMSO was added to dissolve the formazan crystals. The absorbance of each well was measured at 540 nm with an enzyme-linked immunosorbent assay (ELISA) reader (Molecular Devices, Sunnyvale, CA, USA).

After treatment with cordycepin and TRAIL alone or together for the indicated times, the cells were harvested, washed with phosphate-buffered saline (PBS) and fixed with 3.7% paraformaldehyde in PBS for 10 min at room temperature. Fixed cells were washed with PBS, and stained with 2.5 μg/ml DAPI solution for 10 min at room temperature. The cells were washed 2 more times with PBS and analyzed via a fluorescence microscope (Carl Zeiss, Oberkochen, Germany).

The cells were washed twice with cold PBS and then centrifuged. The resulting pellet was fixed in 75% (v/v) ethanol for 1 h at 4ºC. The cells were washed once with PBS and resuspended in cold PI solution (50 μg/ml) containing RNase A (0.1 mg/ml) in PBS for 30 min in the dark. Flow cytometric analyses were performed using FACSCalibur (Becton-Dickinson, San Jose, CA, USA). Forward light scatter characteristics were used to exclude cell debris from the analysis. The sub-G1 population was calculated to estimate the apoptotic cell population.

Caspase activities were determined by colorimetric assays using caspase-3, -8 and -9 activation kits according to the manufacturer’s protocol. The kits utilize synthetic tetrapeptides labeled with p-nitroaniline. Briefly, the cells were lysed in the supplied lysis buffer. The supernatants were collected and incubated with the supplied reaction buffer containing dithiothreitol (DTT) and substrates at 37ºC. Caspase activity was determined by measuring changes in absorbance at 405 nm using an ELISA reader.

Total RNA was prepared using an RNeasy kit and primed with random hexamers to synthesize complementary DNA using AMV reverse transcriptase (Amersham Corp.) according to the manufacturer’s instructions. PCR was carried out in a Mastercycler (Eppendorf, Hamburg, Germany) with the indicated primers. Conditions for the PCR reactions were, 1× (94ºC for 3 min); 35× (94ºC for 45 sec, 58ºC for 45 sec and 72ºC for 1 min); and 1× (72ºC for 10 min). Amplification products obtained by PCR were electrophoretically separated on 1% agarose gels and visualized by ethidium bromide staining.

The cells were harvested and lysed with lysis buffer (20 mM sucrose, 1 mM EDTA, 20 μM Tris-Cl, pH 7.2, 1 mM DTT, 10 mM KCl, 1.5 mM MgCl₂, and 5 μg/ml aprotinin) for 30 min. Protein concentration was measured using a Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions. For western blot analysis, an equal amount of protein was subjected to electrophoresis on SDS-polyacrylamide gels and was transferred by electroblotting to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH, USA). The blots were probed with the desired antibodies for 1 h, incubated with the diluted enzyme-linked secondary antibody, and visualized with an ECL solution according to the recommended procedure.

The MMP of intact cells was measured by DNA flow cytometry with the lipophilic cation JC-1. JC-1 is a rationmetric, dual-emission fluorescent dye that is internalized and concentrated by respiring mitochondria and reflects changes in MMP in living cells. There are two excitation wavelengths. At low values of MMP, JC-1 remains a monomer (FL-1, green fluorescence; 527 nm) whereas it forms aggregates at high MMP (FL-2, red fluorescence; 590 nm) according to the recommended procedure (20). Cells were trypsinized, the cell pellets were resuspended in PBS, and incubated with 10 μM JC-1 for 30 min at 37ºC. The cells were subsequently washed once with cold PBS, suspended and analyzed by flow cytometry.

All data are presented as means ± standard deviation. Significant differences among the groups were determined using the unpaired Student’s t-test. A p-value <0.05 was considered to indicate a statistically significant result. All values were obtained from at least 2 or 3 independent experiments.

We first assessed whether cordycepin sensitizes Hep3B cells to TRAIL-mediated apoptosis. Treatment with 50 ng/ml TRAIL resulted in negligible growth inhibition of Hep3B cells at 24 h (Fig. 1A), suggesting that this cell type is resistant to TRAIL-induced apoptosis. We next examined the cytotoxic effects of cordycepin alone or in combination with TRAIL in Hep3B cells. The concentrations (1–10 μg/ml) of cordycepin alone used in this study did not significantly induce morphological changes or inhibit growth. However, cell viability was significantly inhibited by the co-treatment with cordycepin and TRAIL (Fig. 1A) indicating that the combination of cordycepin and TRAIL substantially induces cell death. To investigate whether decreased cell viability by the combined treatment was due to apoptotic signaling, morphological changes and sub-G1 phase populations of Hep3B cells were determined using DAPI staining and flow cytometric analysis, respectively. As shown in Fig. 1B and C, a significant number of cells co-treated with cordycepin and TRAIL were observed with apoptotic shrinkage, chromatin condensation, loss of nuclear construction and formation of apoptotic bodies, whereas these features were not observed in control cells or cells treated with cordycepin or TRAIL alone. Additionally, treatment of Hep3B cells with a combination of cordycepin and TRAIL significantly increased the accumulation of sub-G1 phase cells, whereas treatment with cordycepin or TRAIL alone did not (Fig. 1D). These results showed that the combined treatment of cordycepin and TRAIL sensitized TRAIL-resistant Hep3B cells to apoptosis.

Mitochondria appear to play a central role in apoptosis. Thus, they have been a major focus of recent studies. The early events that occur during apoptotic cell death are mitochondrial depolarization and loss of pro-apoptotic factors from the mitochondrial inter-membrane space (21,22). As shown in Fig. 2A, treatment of cells with cordycepin or TRAIL alone induced a slight loss of MMP in Hep3B cells; however, combined treatment with cordycepin and TRAIL caused a significant concentration-dependent induction of MMP loss. In particular, the anti-apoptotic Bcl-2 family molecules such as Bcl-2 and Bcl-xL, and IAP family members protect some tumor cell lines from TRAIL-induced apoptosis (22–24); therefore, we investigated the expression levels of members belonging to the Bcl-2 and IAP family. As shown in Fig. 2B and C, the mRNA and protein levels of anti-apoptotic Bcl-2, Bcl-xL, cIAP-1, cIAP-2 and XIAP were reduced in response to treatment with cordycepin plus TRAIL, whereas the levels of pro-apoptotic Bax increased markedly. Collectively, these results indicate that downregulation of Bcl-2 and Bcl-xL, and IAP family member expression and increased loss of MMP are associated with cordycepin-mediated sensitization of Hep3B cells to TRAIL-mediated apoptosis.

Caspases act as important mediators of apoptosis and contribute to the overall apoptotic morphology by cleaving various cellular substrates (24,25). Therefore, we next examined whether caspases were actually activated during cordycepin plus TRAIL-induced cell death in Hep3B cells. As shown in Fig. 3A, western blot analysis revealed that treatment with 5 or 10 μg/ml cordycepin and 50 ng/ml TRAIL alone for 24 h did not significantly decrease proteolytic processing of caspase-3, -8 and -9; however, combined treatment with cordycepin and TRAIL significantly decreased their levels. Furthermore, we found that cleavage of key death substrates indicates activation of effector caspases such as PARP and β-catenin (activated-caspase-3 substrates) (26,27). As a result, combined treatment with cordycepin and TRAIL significantly induced cleavage of PARP and β-catenin in Hep3B cells, whereas treatment with cordycepin or TRAIL alone did not. Additionally, cell lysates containing equal amounts of total protein from cells were assayed to assess in vitro caspase activity. As shown in Fig. 3B, a combined treatment with cordycepin and TRAIL significantly increased caspase-3, -8, and -9 activities in Hep3B cells. These results indicate that co-treatment induces apoptotic death in Hep3B cells, at least in part, through a caspase-dependent pathway.

Recent studies have revealed that phosphoinositide 3-kinase (PI3K)/Akt and the MAPK signaling pathways are important regulators during apoptotic cell regulation as a TRAIL sensitizing signal pathway (28,29). To investigate the roles of Akt and MAPKs in mediating the observed apoptotic response, western blot analysis was used to assess the change in Akt, JNK, ERK, and p38 MAPK phosphorylation. As shown in Fig. 4, treatment with cordycepin decreased only JNK phosphorylation, whereas phosphorylation levels of other kinases, such as Akt, p38 and ERK, did not change. The results also indicate that cordycepin exerted a concentration-dependent effect on JNK dephosphorylation with a fixed concentration of TRAIL. Thus, Hep3B cells were incubated with cordycepin and TRAIL in the presence of SP600125, a well-known JNK inhibitor, to investigate the functional roles of these dephosphorylation events. As shown in Fig. 5A–C, pretreatment with SP600125 markedly increased the morphological changes and condensed chromatin in the nuclei, and the number of cells with sub-G1 DNA content. Consistent with the increase in sub-G1 DNA content, pretreatment with SP600125 significantly increased the growth inhibition induced by the combined treatment (Fig. 5D). These results indicate that the combined treatment with cordycepin and TRAIL triggered the inhibition of JNK activation; therefore, we verified the inhibition of JNK signaling responsible for the TRAIL-sensitizing effect of cordycepin on apoptosis in Hep3B cells. As a result, SP600125 enhanced upregulation of pro-apoptotic protein Bax levels and downregulation of anti-apoptotic Bcl-2, Bcl-xL and IAP family protein levels in TRAIL and cordycepin-treated Hep3B cells (Fig. 6A). Moreover, SP600125 enhanced caspase activities, as well as PARP and β-catenin cleavage under the same experimental conditions. Taken together, these findings suggest that the JNK signaling pathway acts as a key regulator of apoptosis in response to the combined cordycepin and TRAIL treatment in Hep3B cells.