Cordycepin Induced MA-10 Mouse Leydig Tumor Cell Apoptosis through Caspase-9 Pathway

In the present study, the apoptotic effect of cordycepin on MA-10 cells, a mouse Leydig tumor cell line, was investigated. Results demonstrated that the number of rounding-up cell increased by cordycepin (10 μM to 5 mM for 24 h), and cells with plasma membrane blebbing could be observed by 100 μM cordycepin. In viability test, MA-10 cell surviving rate significantly decreased as the dosage (10 μM to 5 mM) and duration (3–24 h) of cordycepin treatment increased (P < 0.05). Cordycepin at 100 μM and 1 mM for 24 h treatment induced significant DNA fragmentation (P < 0.05). In addition, the percentage of G1 and G2/M phase cell significantly declined by cordycepin (100 μM and 1 mM) for 24 h treatment, while the percentages of subG1 phase cell increased by 100 μM and/or 1 mM cordycepin in 6, 12 and 24 h treatments (P < 0.05), respectively, which highly suggested that cordycepin induced MA-10 cell apoptosis. In mechanism study with the treatments of caspases, c-Jun NH2 terminal kinase (JNK) or reactive oxygen species (ROS) inhibitors plus cordycepin for 24 h, only caspases inhibitor suppressed subG1 phase in MA-10 cells. Moreover, western blotting results showed that cordycepin induced caspase-9, -3 and -7 protein expressions, but not caspase-8, in time- and dose-dependent manners. In conclusion, cordycepin induced apoptosis in MA-10 mouse Leydig tumor cells through a caspase-9 and -3 and -7 dependent pathway.


Introduction
Cordycepin (3 deoxyadenosine), the analog of adenosine, is a constituent from the mycelia of Cordyceps sinensis (CS), and was considered as an active component with effects altering cytokines secretion, improving lung function, increasing energy levels and sex drive [1][2][3]. It has been demonstrated that cordycepin has anti-tumor effect on mouse melanoma and lung carcinoma cells and human oral cancer cells [4,5]. Moreover, cordycepin could inhibit polyadenylate polymerase (PAP) or inactivate mRNA polyadenylation to induce tumor cell apoptosis, which is characterized by the cellular rounding-up, cytoplasmic contraction, plasma membrane blebbing, chromatin condensation, DNA fragmentation and many biochemical characteristics [6][7][8][9][10][11]. However, the molecular mechanisms regarding apoptotic signal pathways remain elusive.
The activation of cystein aspartic-specific proteases (caspases) is commonly thought to be one of the earliest points in the no-return pathway of apoptosis. Caspases are broadly categorized into upstream regulatory caspases and downstream effector caspases [12]. The upstream caspases, such as caspase-8 (death receptor pathway) and caspase-9 (mitochondria pathway), typically have a long N-terminal prodomain that facilitates interaction with and recruitment of proapoptotic proteins, including other caspases [13]. The downstream caspases, such as caspase-3, -6, and -7, typically have short prodomains that primarily cleave protein, which is important for cellular functions, and results in cell apoptosis [9,[14][15][16]. Moreover, some investigations have indicated that c-Jun NH 2 terminal kinase (JNK) pathway also participates in apoptosis. JNK, a stress-activated protein kinase, is a subgroup of the MAPK superfamily, which can be activated by cell stress such as ultraviolet, TNF and interleukin-1 [17,18]. Furthermore, reactive oxygen species (ROS), molecules possessing an odd of electrons, could induce various biological responses, including cell growth, arrest and/or cell damage [19,20]. Excess ROS would cause damage to cellular component such as lipid membranes, protein, and DNA, leading to apoptosis [21][22][23].
We have previously demonstrated that CS could induce MA-10 cell apoptosis [24]. It is possible that cordycepin, 2 Evidence-Based Complementary and Alternative Medicine the pure substance from C. sinensis, induce MA-10 cell death. Although some reports have demonstrated that cordycepin possesses anti-tumor action, limited reports about cordycepin-mediated apoptosis in testicular tumor cells have been illustrated. In addition, it has been demonstrated that between 1973 and 1995 the incidence of testicular cancer in the United States increases 51% (1% of total cancer cases annually), and orchidectomy is the common protocol to treat testicular and Leydig cell cancers [25,26]. Thus, rather than orchidectomy, the exploration of cordycepin-induced cell death with the study of mechanism will be valuable to design more effective chemotherapy agents on testicular tumor cells.

Cell
Culture. The MA-10 cell line was a gift from Dr Mario Ascoli (University of Iowa, Iowa City, IA, USA), and was maintained with the standard technique [27]. Cells were maintained in Waymouth medium containing 15% FBS and incubated in a humidified atmosphere containing 95% air and 5% CO 2 at 37 • C.

Morphology
Observation. MA-10 cells (6 × 10 5 ) were seeded in 6-cm Petri dish (Techno Plastic Products AG, Trasadingen, Switzerland) with 2 mL serum medium. After 70−80% confluence, cells were treated without or with 10 μM, 100 μM, 1 mM, 2 mM and 5 mM cordycepin for 24 h. Cell morphology was then observed and recorded under light microscopy (Olympus, CK 40). Apoptosis is characterized by the loss of cellular contact with the matrix and the appearance of plasma membrane blebbing [9].

DNA Fragmentation Assay.
In order to investigate if cordycepin could induce cell apoptosis, DNA fragmentation was determined first. After treatment, MA-10 cells (1 × 10 6 ) were lysed in a 0.6 mL cell lysis solution containing 20 mM Tris−HCl, 10 mM EDTA, pH 8.0 and 0.3% Triton X-100. DNA was extracted with 0.6 mL phenol/chloroform (1 : 1), and the mixture was centrifuged at 12 500 r.p.m. for 10 min. DNA in the aqueous phase was extracted with phenol/chloroform (1 : 1) again. The aqueous phase with DNA was mixed with isopropanol at −20 • C overnight. After centrifugation, DNA pellets were washed with 70% ethanol and air-dried. DNA pellets were dissolved in TE buffer (10 mM Tris−HCl, 1 mM EDTA, pH 8.0), and RNase A (3 mg/mL) was added to remove RNA at 37 • C for 30 min. DNA electrophoresis was carried out in 2% agarose gel. The gel was stained with ethidium bromide. DNA fragments were visualized under UV light and photographed.

MTT Cytotoxicity Test.
Methylthiazoletetrazolium test was used to determine cell viability with the treatment of cordycepin [28]. MA-10 cells were seeded in 96-well plate (Techno Plastic Products AG, Trasadingen, Switzerland) containing 2 × 10 4 cells with 100 μL serum medium in each well. After 70−80% confluence, cells were treated without or with 10 μM, 100 μM, 1 mM, 2 mM and 5 mM cordycepin for 3, 6, 12, and 24 h, respectively. MTT was added at different time points with the final concentration of 0.5 mg/mL, and then incubated at 37 • C for 4 h. The medium was removed and DMSO (50 μL) was added into each well to dissolve the crystals by shaking the plate weakly for 20 min in dark. The OD values in each treatment were then determined at λ = 590 nm by an ELISA reader (Opsy MR, Dynex, USA).

Flow Cytometry Analysis.
To further confirm whether cordycepin could induce cell apoptosis, the redistribution of cell cycle by flow cytometric analysis was used with propidium iodine stain [29,30]. MA-10 cells (6 × 10 5 ) were seeded in 6-cm dish with 2 mL serum medium. After 70−80% confluence, cells were treated with free medium containing various concentrations of cordycepin for 3, 6, 12, and 24 h, respectively. Cordycepin-treated cells were harvested with trypsin, washed with PBS, and mixed in 75% ethanol for at least 2 h at −20 • C. After fixation, cells were washed with cold PBS and then collected by centrifugation, mixed with 100 μg/mL RNase, and stained with PI (propidium iodide) solution containing 40 μg/mL in PBS. The stained cells were analyzed using a fluorescence activated cell sorter cycle distribution, called hypodiploid. The hypoploid DNA contents represent the DNA fragmentation [30].

Immunoblotting
Analysis. Cells (6 × 10 6 ) were seeded in 6-cm dish. After treatment, cells were rinsed with cold PBS. Cells were then harvested by 100 μL lysis buffer (50 mM Tris-base, 150 mM NaCl, 1% NP40, 0.1% SDS, 0.5% deoxychloride acid and 1 mM PMSF). Cell lysate was centrifuged at 32 000 r.p.m. for 10 min at 4 • C. The pellet was collected by 10 μL lysis buffer and was centrifuged again at 12 000 g. The supernatant, which contains total protein, was collected and stored at −20 • C. Protein concentration of the cell lysates were determined by the Lowry method [31]. Cell proteins (40−60 μg) were separated in 12% SDS−polyacrylamide gel, that performed at 100 V for 2.5 h using standard running buffer ( buffer (20 mM Tris−HCl, 150 mM glycine, 10% methanol, 0.01% SDS). The membranes were blocked with 5% nonfat milk, washed by TBST (20 mM Tris-base, 137 Mm NaCl, 0.1% Tween 20, pH 7.6), and subsequently incubated with primary caspase-3 or -7 antibodies at 1 : 4000 dilutions and caspase-8 or -9 antibodies at 1 : 2000 dilutions overnight at 4 • C. After washing, the membrane was incubated with horseradish peroxidase-conjugated sheep anti-mouse antibody or donkey anti-rabbit antibody, and then visualized by enhanced chemiluminescence (ECL) detection kit (Amersharn-Pharmacia International PLC, UK). The optical density of each protein band was quantitated by a Quantity One (PDI, Huntington Station, NY, USA) computer-assisted image analysis system [32]. The amount of β-actin in each lane was also detected as a control to correct the expression of caspases proteins.

Statistic Analysis.
Each data point in the figures represents the mean ± SEM of three separate experiments. Statistically significant differences between treatments and controls were determined by one-way ANOVA and then Least Significance Difference (LSD) comparison procedure. Statistical significance was set at P < .05.

The Involvement of Cordycepin on ROS, JNK and/or
Caspase Pathways Related to Apoptosis in MA-10 Cells. To investigate possible pathway that cordycepin might activate to induce MA-10 cell apoptosis, cells were co-treated with 100 μM cordycepin plus 1 μM JNK inhibitor (SP600125), 1 mM ROS inhibitor (NAC) or 100 μM general caspase inhibitor (zVAD-fmk) for 24 h. The change of subG1 ratios determined by flow cytometry was then analyzed. Figure 5(a) illustrates that subG1 ratio increased from 2.97% in control group to 39.82% in 100 μM cordycepin treatment after 24 h (P < .05). JNK and ROS inhibitors could not suppress the subG1 ratio (40.76 and 40.05%, resp.) back to control levels (P > .05). In caspase inhibitor experiment ( Figure 5(b)), the subG1 ratio increased from 1.78% in control group to 14.66% in cordycepin treatment after 24 h (P < .05). Interestingly, general caspase inhibitor significantly decreased subG1 ratio to 9.26% (P < .05). These results indicated that caspase activation/pathway played roles in cordycepininduced apoptosis in MA-10 cells.

Discussion
In the present study, cordycepin would induce MA-10 cell rounded-up with blebbed membrane, and reduced MA-10 cell viability with apparent DNA fragmentation. It is well known that internucleosomal DNA fragmentation displays as a biochemical hallmark during apoptosis [33]. The induction of DNA fragmentation phenomenon by cordycepin at 100 μM for 24 h correlated with the increase of subG1 phase cell number of cytometry analysis, which confirms that cordycepin could induce apoptosis in MA-10 cells. Meanwhile, there were significant decreases of G1 and G2/M phase cells. Moreover, caspases inhibitor, but not JNK and ROS inhibitors, decreased subG1 ratio, implying that caspase pathway played role in cordycepin-induced apoptosis. Furthermore, cordycepin induced caspase-9, -3 and -7 expressions, but not caspase-8, to activate MA-10 cell apoptosis.
Hyperphosphorylation of PAP from p34 cyclin B kinase during mitosis will lead to the inactivation of PAP, which contributes to the reduction of mRNA and protein synthesis during the M phase of the cell cycle [34]. Also, PAP activity levels are significantly elevated at the G1/S phase of the cell cycle, along with the increased rate of mRNA polyadenylation and accumulation in the cytoplasm [35]. In the present study, treatment of MA-10 cells with cordycepin lead to the decreases of G1 and G2/M phases. It is possible that cordycepin inhibited polyadenylation, which was necessary at the G1/S phase of the cell cycle in order for all the required mRNAs to be polyadenylated, transferred to the cytoplasm and translated, in order for mitosis to take place. As a result, mitosis will not finish, and cells will arrest at the onset of apoptosis, since not all necessary proteins are translated. Indeed, the decreases of G1 and G2/M phases may imply that MA-10 cells were very sensitive to cordycepin since there was no G2/M phase arrest.
Many evidences have demonstrated that excess ROS in cell could induce cell apoptosis [22,23,36,37]. An increase in ROS is also associated with the activation of redox sensitive JNK/MAPK signaling pathway, which is often involved in the transcriptional activation of genes and posttranslational modifications of proteins necessary for apoptosis [23,38]. Hence, in the present study, both JNK and ROS inhibitors should be able to inhibit apoptotic effect. Interestingly, JNK inhibitor and ROS inhibitor could not decrease the subG1 ratio, which indicated that both JNK and ROS do not play any role in cordycepin-induced apoptosis in MA-10 cells. However, caspase inhibitor did suppress the subG1 ratio, which highly suggested that caspase pathway was involved in the cordycepin-induced apoptosis in MA-10 cells. Many investigations have illustrated that caspases play essential role in apoptosis [39]. Thus, our observations are not unprecedented.
In the present study, 100 μM and 1 mM cordycepin behaved differently to induce temporal expression of caspases. In 100 μM cordycepin treatment, the maximal expression of caspase-3 and -7 were at 6 and 12 h, respectively. Although there were increasing trends of caspases-8 and -9 expressions at 12 h, no significances were observed. This phenomenon illustrated that there was no caspase initiator to activate effector caspase-3 and -7. It has been shown that other initiators, such as caspase-2 and/or caspase-10 can activate effector caspases [40,41]. It is possible that 100 μM cordycepin could induce caspase-2 and/or caspase-10 to activate caspase-3 and -7 expressions for cell apoptosis. In 1 mM cordycepin treatment, caspase-9 expression (the initiator) significantly increased first at 12 h, and then caspase-3 (the effector) increased at 24 h, which illustrated a logical temporal phenomenon to induce MA-10 cell apoptosis. The possible mechanism of how different dosages of cordycepin regulating MA-10 cell apoptosis will be interesting to be further investigated.
In general, the induction of caspase-9 is due to the activation of mitochondrial apoptotic pathway, which can be activated by cytochrome c, ROS and JNK pathway and/or caspase-2 to induce cell apoptosis [42,43]. In the present study, we observed that caspase-9 was activated without any involvement of ROS and JNK. It is possible that cordycepin could induce cytochrome c, caspase-2 or other alternative pathways to induce caspase-9 expression in MA-10 cells. This indecision will be further examined.
It has been shown that cordycepin is an analog of adenosine. The possibility of adenosine receptor involved in apoptotic effect on some types of cancer cell is also proposed [44]. Thus, it is possible that cordycepin might associate with adenosine receptor to activate apoptosis in MA-10 cells. Future works regarding the investigation on the relations to adenosine receptor in MA-10 cells will be valuable.
In conclusion, cordycepin could induce the decrease of G1 and G2/M cell numbers and the increase of subG1 cell number, followed by significant apoptotic cell death in MA-10 mouse Leydig cell line. The expressions of caspases also demonstrate that cordycepin did induce apoptosis on MA-10 mouse Leydig tumor cell line. Many Chinese herbs and its pure compounds have been demonstrated with the anticancer effects [45,46]. This promising observation about the apoptotic effect of cordycepin on testicular cancer cell may be considered as a good approach in search and development of new anti-cancer drug.