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Synergistic property of cordycepin in cultivated Cordyceps militaris-mediated apoptosis in human leukemia cells.


Cordyceps militaris is a well-known Chinese traditional medicinal mushroom frequently used for tonics and recently of a potential interest for cancer intervention. Here, we explored the cancer cell killing activity of the hot water extracts of C. militaris cultured mycelia ([CM.sub.MY]) and cultivated fruiting bodies ([CM.sub.FB]). We found that [CM.sub.FB] exhibited a greater cytotoxic effect against various cancer cells over [CM.sub.MY]. Apoptotic phenotypes including apoptotic body formation, DNA laddering, caspase 3 activation and cleavage of PARP proteins were induced by [CM.sub.FB] treatment but only slightly induced by same concentration of [CM.sub.MY] treatment in human HL-60 leukemia cells. Cordycepin in [CM.sub.FB] (10.47 mg/g) is significantly higher (~15.2 times) than that of [CM.sub.MY] (0.69 mg/g). Using isobolographic analysis, the synergy of cytotoxicity was observed across different combined concentrations of [CM.sub.MY] and cordycepin. By complementing cordycepin into [CM.sub.MY] to the level comparable with CMFB, we observed that [CM.sub.MY] (500 [micro]g/ml) with cordycepin (4.8 [micro]g/ml) induced apoptosis to a level similar to that induced by [CM.sub.FB] (500 [micro]g/ml). Together, our results suggest that cordycepin possesses a synergistic cytotoxic effect with Cordyceps militaris-mediated apoptosis in human leukemia cells and therefore explaining a better anti-proliferating activity of [CM.sub.FB] over [CM.sub.MY].


Cordyceps militaris





Cancer-cell killing


Cordyceps militaris, like Cordyceps sinensis, belongs to Ascomycetes and they parasitize insects at the larval stage and gradually grow into fruiting body expanded outside the insect larvae or pupae (Buenz et al., 2005). This parasitic complex of fungus and insect has long been used for tonics and folk medicinal purposes, especially in East Asia (Stone, 2008, 2010). It is commonly used for anti-aging (Das et al., 2010; Ji et al., 2009; Li et al., 2010) and also has been used for the treatments of various diseases such as hypertension (Ahmed et al., 2012), arrhythmia (Yan et al., 2013), hepatitis (Niwa et al., 2013) and hyperglycemia (Kan et al., 2012; Lo et al., 2004,2006). Interestingly, recent reports have suggested a potential anti-tumor activity (Das et al., 2010; Reis et al., 2013; Yang et al., 2006).

Due to the rarity of C. sinensis, C. militaris has been regarded as a substitute for C. sinensis (Das et al., 2010). Although pharmacologically active components remain largely unresolved, experimental results reveal that C. militaris has a similar chemical composition to C. sinensis including cordycepin (3'-deoxyadenosine), D-mannitol (cordycepic acid) and polysaccharides (Lim et al., 2012). It should be noted here that cordycepin, an inhibitor of RNA polymerases, is considered as one potential pharmacological ingredient of Cordyceps spp. for anti-tumor activity (Chen et al., 2008; Choi et al., 2011; Koc et al., 1996; Tuli et al., 2013).

Not until recently, it is not possible to cultivate C. militaris in vitro, especially in the form of fruiting body (Das et al., 2010; Shrestha et al., 2005). The availability of large quantity of cultivated C. militaris has allowed scientists to investigate its biologically active ingredients responsible for various medicinal usages (Das et al., 2010).

In this present study, both mycelia and fruiting bodies were produced from laboratory cultivation. The hot water extracts of mycelia ([CM.sub.MY]) and fruiting bodies ([CM.sub.FB]) were prepared for biological study. Our results showed that [CM.sub.FB] exhibited a much greater cytotoxic activity against various cancer cell lines than that of [CM.sub.MY]. To investigate the underlying mechanism(s) giving rise to differential cytotoxic abilities of [CM.sub.FB] and [CM.sub.MY]. induced cell-death pathways and corresponding chemical compositions were analyzed. Consistently, [CM.sub.FB] also displayed a better apoptosis-inducing activity than that of [CM.sub.MY]. HPLC analyses revealed that [CM.sub.FB] contains a 15-fold higher concentration of cordycepin compared to that of [CM.sub.MY]. The isobolographic analysis defined a synergistic interaction between cordycepin and [CM.sub.MY]. The reconstitution experiments further showed that a comparable administration of cordycepin (4.8 [micro]g/ml, <10% apoptotic cells induced) into [CM.sub.MY] (500 [micro]g/ml, <10% apoptotic cells induced) synergistically promoted the apoptosis-inducing capability to the level of [CM.sub.FB] (500 [micro]g/ml, ~80% apoptotic cells). In conclusion, our results suggested that the synergistic apoptosis-inducing ability of cordycepin with C. militaris extract might provide a plausible explanation for the greater anti-cancer activity of fruit bodies over mycelia.

Materials and methods

Cultivation of Cordyceps militaris

C. militaris was obtained from Mucho Biotech Co. (Taipei, Taiwan). The composition of culture medium for producing fruiting bodies was 70% (w/w) rice, 23% (w/w) silkworm chrysalis powder, 5% (w/w) sucrose, 1.5% (w/w) peptone and 0.5% (w/w) yeast extract. Thirty grams of the above components were dispensed into bottles containing 30 ml of water. The bottles were wrapped with polypropylene film before being autoclaved. Each bottle was inoculated with 3% (v/v) of C. militaris. The culture condition was 15-20[degrees]C and 65% of humidity in a dark environment. When mycelia covered the entire bottle surface and proliferated to the bottom of the bottle, fruiting body development was stimulated by exposure light for 12 h a day at 85% of humidity and 20-25[degrees]C. The formation of fungal orange buds was observed at the 14th day. When the fruiting body grew to a height of 8 cm, it could be harvested for extraction. The isolated mycelia from C. militaris for the bioassays were transferred to seed culture SDAY plates containing Sabouraud dextrose agar (SDA) and yeast extract by punching out about 6 mm diameter agar disks from culture grown on potato dextrose agar (PDA) plates and maintained at 25[degrees]C in the dark for 14 days.

Extract preparation and HPLC analysis

The collected fruiting body or mycelium of C. militaris was dried at 50[degrees]C and homogenized in liquid nitrogen with a pestle. One gram of sample powder was dissolved in distilled water with a ratio of solid: liquid of 1: 40 and vortexed for 30 s. The mixture was placed in a water bath with a constant temperature of 50[degrees]C for 2 h and sonicated (power 150 W) for the first 30 min. The liquid phase was separated from the solid by centrifugation at 3500 x g for 20 min and then filtered with 0.22 nm filter.

For HPLC analysis, the mobile phase was methanol: water (80:20, v/v) and the flow rate was 1.0 ml/min. For determination of adenosine, D-mannitol and cordycepin, a Purospher Lichro CART PP18 column (Merck) was used at 40[degrees]C. The UV detection was at 260 nm, and the injection volume was 20 [micro]l. The external standard method was applied. Identification of targeted components was compared with their retention time and spectrum against known standards.

Cell culture and MTT assay

The HCT116 colorectal and HL-60 leukemic cancer cell lines were a generous gift from Dr. L.F. Liu (Univ. Med. Den., NJ, USA). The PC-3 prostate and Huh7 liver cancer cell lines and BJ normal skin and Wl-38 lung fibroblast cell lines were from Dr. T.-L. Shen (National Taiwan Univ., NTU, Taiwan). The other of all cell lines except HL-60 were grown in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) at 37[degrees]C and a 5% C[O.sub.2] atmosphere. The HL-60 cells were grown in RPMI Media 1640 (Gibco) medium.

Viability of control and treated cells was evaluated using MTT assay in triplicate. Cells of dimension 1 x [10.sup.3] (per well) were seeded in 96-well plates containing 100 [micro]l culture medium per well at 24 h before treatment. After 4 days of treatment with the appropriate concentrations of the hot water extract of Cordyceps militaris, the cells were incubated at 37[degrees]C in 200 [micro]l MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (Sigma) solution (5 mg/ml) for 4 h. After removal of the medium and MTT, 100 [micro]l of DMSO was added to each well for optical density (OD) reading at 570 nm using an ELISA reader (Model 680, Bio-Rad). The percent viability was calculated as (test OD/control OD) x 100.

Apoptotic bodies formation assay

For detection of apoptotic morphology and chromatin condensation, the cells were fixed after treatment with 100% methanol at -20[degrees]C for 10 min and then washed twice with 1 x PBS. The cells were stained with 10 pM of Hoechst 33342 (Sigma) in 1 x PBS for 1 h and washed two more times with 1 x PBS. Apoptotic morphological and nuclear changes were scored under an epi-fiuorescence microscope (Nikon Eclipse 80i) with a CCD camera (Nikon DS-Ri1). Up to 200 stained cells per field were captured to examine the formation of apoptotic bodies.

DNA fragmentation assay

The fragmented DNA extract was prepared from the treated HL-60 cells. After 12 h treatment, the cells were lyzed with buffer A (1% NP-40, 20 mM EDTA, 50 mM Tris-HCl pH 7.5) and then subjected to centrifuge at 2500 x g for 5 min to acquire the supernatant fraction containing the fragmented DNA. SDS (final cone. 1%) and RNase A nuclease (final 5 [micro]g/pl) were added into the supernatant for 1 h at 56[degrees]C prior to adding the proteinase K (final 2.5 [micro]g/pl) at 37[degrees]C for another 1 h. After ethanol precipitation, 1.5% agarose electrophoresis was performed to analyze the DNA fragments.

Immunoblotting analysis (Western blotting)

The [CM.sub.MY] or [CM.sub.FB] treated HL-60 cells were lyzed with 1x sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 1% [beta]-mercaptoethanol and 0.02% bromophenol blue). The collected samples were resolved by SDS-PAGE on 8% gels, and then transferred to nitrocellulose membranes (Pall) at 330 mA for 2.5 h at 4[degrees]C. The membranes were blocked with 5% non-fat milk in 1 x TBS (Tris buffered saline, 50 mM Tris-HCl pH 7.4 and 150 mM NaCl) for 1 h at room temperature. The membranes were then washed two times with 1 x TBS and incubated with the primary antibody against PARP (Cell Signaling), cleaved caspase 3 (Cell Signaling) or GAPDH (Cell Signaling) for 12 h at 4[degrees]C. The membranes were then washed three times with 1 x TBS containing 0.2% Tween 20 and then incubated with horseradish peroxidase conjugated anti-rabbit or anti-mouse IgG antibody for 1 h at room temperature. The membranes were finally washed three times with 1 x TBS containing 0.2% Tween 20, and then subjected to enhanced chemiluminescence detection (PerkinElmer Life Science). Antibodies for GAPDH were used as the internal loading standard. Immunoblotting results were scanned and saved as electronic files. The intensities of individual bands were determined with the ImageQuant program (Molecular Dynamics).

Isobolographic analysis to cellular survival

To describe the dose-dependent interaction of [CM.sub.MY] and cordycepin, isobolograms at effective levels of 50% inhibition ([IC.sub.50]) of leukemia cell proliferation were performed. Since the single agents --alone or in combinations--usually reached 50% cancer cell inhibition, the 50% isobologram provided an actual comparison of the single use versus the combinations (Wagner and Ulrich-Merzenich, 2009). In each of these, additivity was determined by extrapolating the dose requirements for each extract or compound in combinations from its single use ([IC.sub.50]). The effects of drug combinations were examined by statistical analyses. The resulting iso-effect curve of [IC.sub.50] was given to depict the synergistic effect of the cordycepin-CMMY interaction. Data points above or below the line of additivity indicate antagonism or synergy, respectively.


Statistical analyses

All of MTT assay, apoptotic bodies formation assay and DNA fragmentation assay had three repeated and was performed with Student's t-test for statistical analysis. Data were considered statistically significant when p-values versus control were [less than or equal to] 0.05 and [less than or equal to] 0.01, which are shown with single (*) and double asterisks (**) respectively.

Results and discussion

Cultivation of CM and preparation of its extracts

We have established a procedure to produce C. militaris fruiting bodies using a solid rice-based medium inoculated with liquid spawn as shown in Fig. 1A. After cultivation at 25[degrees]C in a humid atmosphere (85%), C. militaris showed increased fruiting body mass from 14 to 43 days (Fig. IB). At the 14th day, there were some light orange buds emerging from the rice medium under appropriate stimuli such as exposure to light as well as increase in humidity and temperature, indicating that a transformed stage from the mycelium to the fruiting body. Upon the growth of fruiting body to a height of approximately 8 cm (55 days of cultivation), it could be harvested for extraction. For mycelium production, inoculation was on the SDAY culture medium for 14 days in the dark at 25[degrees]C as shown in Fig. 1A and C. For preparation of the water extracts of CM applied in this study, the fruiting body and the mycelium were dried and homogenized in liquid nitrogen to form a powder, separately. The water extracts of fruiting bodies ([CM.sub.FB]) and mycelia ([CM.sub.MY]) were then prepared by dissolving the powder in hot water at 50[degrees]C and subsequently being centrifuged, as described in the Materials and methods.

[CM.sub.FB] treatment results in more growth inhibition in various kinds of cancer cells compared to [CM.sub.MY]

We then assessed the effect of [CM.sub.FB] and [CM.sub.MY] on human cancer cell death. The human colorectal FICT116, prostatic PC-3, hepatic Huh7 and leukemic HL-60 cancer cell lines were treated with increasing concentrations of [CM.sub.FB] or [CM.sub.MY] for 4 days and then subjected to MTT analysis. As shown in Fig. 2A, treatments with the low doses of 7.8-125 [micro]g/ml of [CM.sub.FB] and [CM.sub.MY] resulted in a significant decrease in cell viability in all of tested cancer cell lines. In all cancer cell lines, [CM.sub.FB] reduced cell viability more effectively than [CM.sub.MY]. In contrast, [CM.sub.MY] or [CM.sub.FB] exhibited an insignificant influence on cell survival in the two normal human cell lines (BJ and WI-38)(Fig. 2B).


[CM.sub.FB] triggers more cellular programmed death in leukemia HL-60 cells than [CM.sub.MY]

To reveal the medicinal activity of Cordyceps militaris, we have found the water extract of this fungus might provide a low toxicity but great efficacy of anti-cancer effect in a broad list of human cancers including colon cancer, prostate cancer, liver carcinoma and leukemia. Since apoptosis has been considered as an important mechanism for cancer therapies, we examined whether the programmed cell death is promoted and how it proceeds after treatment of [CM.sub.FB] and [CM.sub.MY] by performing program cell death assays. First, the apoptosis-related morphological alterations of HL-60 cells treated with or without [CM.sub.FB] and [CM.sub.MY], respectively, were stained with Hoechst 33342 and examined using fluorescent microscopy. Our results indicated that [CM.sub.FB], compared to [CM.sub.MY], more efficiently led to cancer cell apoptosis as obviously shown by cell shrinkage, chromatin fragmentation and the loss of normal nuclear architecture (Fig. 3A and C). The quantified results of apoptotic body formation and DNA laddering were shown in Fig. 3B and D, and the amount of VP-16-induced apoptotic bodies and DNA ladders was considered as 100%. Accordingly, although the formation of apoptotic bodies slightly increased at the dose of 2000 [micro]g/ml, low doses of [CM.sub.MY] did not induce cell apoptosis similar to the non-treatment control. The [CM.sub.FB]-triggered DNA fragmentation was 81.2% at 500 [micro]g/ml of [CM.sub.FB] in contrast to that [CM.sub.MY] gave rise to a slight effect (12.4% at 2000 [micro]g/ml of [CM.sub.MY]).

To determine whether the caspase pathway is responsible for the induction of leukemia cell apoptosis upon [CM.sub.FB] or [CM.sub.MY] treatment, the cell lysates were collected and subjected for the immunoblotting assay to detect the cleaved PARP (an apoptotic indicator) and the cleaved caspase 3. As the matter of fact the cleavage of native caspase 3 (32 kDa) into a 17 kDa and an 11 kDa occurs upon cell apoptosis, which in turn further cleaves PARP by the activated caspase 3. We found that the [CM.sub.FB] significantly elicited caspase 3 activation and then led to the cleavage of PARP in a dose dependent manner. In contrast, there was little caspase 3 activation by [CM.sub.MY] treatment. Our results suggested that [CM.sub.FB] enables to induce apoptosis in the leukemia FIL-60 cells much stronger than [CM.sub.MY] does.


Cordycepin but not D-mannitol or adenosine induces apoptosis in leukemic HL-60 cells

The discrepancy on apoptosis between [CM.sub.FB] and [CM.sub.MY] as described above was investigated. First, we examined the contents of well-known bio-constituents in Cordyceps spp., including adenosine, D-mannitol and cordycepin by HPLC analyses. Representative results were shown in Fig. 4A and B. [CM.sub.FB] contained more than 15 times amount of cordycepin compared to [CM.sub.MY] (Fig. 4D). By contrast, the contents of adenosine and D-mannitol in [CM.sub.MY] were even greater than those in [CM.sub.FB]. Recent research has shown that cordycepin (Fig. 4C), 3'-deoxyadenosine, a unique compound exclusively purified from Cordyceps spp., is an inhibitor of polyadenylation associated with the stability of mRNA and cell survival (Imesch et al., 2011). Nevertheless, cordycepin is also attractive on its role for inhibiting platelet aggregation (Cho et al., 2007), bearing anti-inflammatory (Kim et al., 2011) and anti-cancer effects (Tuli et al., 2013). In fact, anti-cancer properties of cordycepin have been studied in several types of cancer cells (Baik et al., 2012; Lee et al., 2012, 2013).


Therefore, we tested whether cordycepin leads to cancer cell death due to induction apoptosis elucidated by DNA fragmentation assay as well as immunoblottings of the cleavage products of caspase 3 and PARP in HL-60 cancer cells. Cordycepin (25 [micro]g/ml) resulted in an increase of DNA fragmentation compared to the control treatment (Fig. 5A). The quantified results (Fig. 5B) indicated that 100 [micro]g/ml of cordycepin induced 99% of the fragmentation similar to VP-16 did. However, neither adenosine nor D-mannitol caused apoptosis in HL-60 cells, even at high concentration of 100 [micro]g/ml. Moreover, only cordycepin enabled activation of caspase 3, as indicated by the cleavage and activation of caspase 3 and PARP (Fig. 5C). Taken together, our data showed by DNA fragmentation assay or immunoblotting assay against the cleaved PARP and caspase 3 suggest that only cordycepin and not adenosine or D-mannitol exhibits potential to induce apoptosis in treated cells (Fig. 5).

Synergistic effect of anti-proliferation by use of [CM.sub.MY] and cordycepin in combination

Next, the anti-proliferation effects of [CM.sub.MY] and cordycepin in combination were evaluated using MTT assay. First, we used the consistent dose (20 [micro]g/ml) of [CM.sub.MY] mixed with various dosages of cordycepin for 4 days of treatment in HL-60 leukemia cells. The collected results were shown in Fig. 6A. The cell survival rate was decreased by cordycepin in a dose dependent manner compared with [CM.sub.MY] alone indicating that cordycepin has a property to elevate the ability of [CM.sub.MY] to down-regulation of cell viability. Further, to determine whether a synergistic interaction between [CM.sub.MY] and cordycepin, isobolographic analysis was carried out as this method has been used to determine whether the combination of drugs leads to a synergistic, additive or antagonistic effect (Berenbaum, 1989; Wagner and Ulrich-Merzenich, 2009). Through various dosages of combinations of [CM.sub.MY] and cordycepin, the concave shape of the resulting 50% isobole for inhibition of cell proliferation (IC50) was displayed in Fig. 6B. Based on H. Wagner and G. Ulrich-Merzenich's review, the interaction index is <1 and, therefore, corresponds to the isobole that is concave curve toward to the zero point. The result indicates that the combination of [CM.sub.MY] and cordycepin exerts a synergistic effect on the inhibition of cell viability of HL-60 leukemia cells.

Cordycepin is a major factor in induction of apoptosis

Furthermore, we also tested whether CMmv could exhibit the same induction of apoptosis as [CM.sub.FB] upon that the content of cordycepin in [CM.sub.MY] is evaluated to a comparable amount same as in [CM.sub.FB]. Accordingly, approximatly 4.8 [micro]g/ml cordycepin was administrated into 500 [micro]g/ml of [CM.sub.MY] to make up to the concentration of cordycepin at 5.2 [micro]g/ml similar to that in [CM.sub.FB] before subjected to DNA fragmentation assay and immunoblotting. As expectedly, the intensity of DNA laddering (Fig. 7A) and the amounts of cleaved PARP and caspase 3 (Fig. 7C) were elevated in the treatment of [CM.sub.MY] with [CM.sub.FB]-comparable cordycepin compared to [CM.sub.FB] alone. Statistically (Fig. 7B), by comparing with VP-16-mediated DNA laddering, the treatment of [CM.sub.MY] with [CM.sub.FB]-comparable cordycepin caused approximate 79% of DNA laddering similar to the [CM.sub.FB] treatment (85%), suggesting that cordycepin is indeed the one of major culprits to induce apoptosis. Interestingly, 5.2 [micro]g/ml of cordycepin alone was not sufficient to trigger apoptosis in HL-60 cells (Fig. 7B and C).


In conclusion, the results of this study have shown that a broad anti-cancer effect of [CM.sub.FB] and [CM.sub.MY] from the cultivated C. militaris in our laboratory as well as illustrated that cordycepin is a crucial constitute with a synergistic property in the water extract of C. militaris leading to induction of cancer cell apoptosis. The relevance for cordycepin rather than D-mannitol or adenosine involving in induction of cancer apoptosis comes from the observation that less the cordycepin, the less apoptosis. Nevertheless, we did not find cordycepin alone with the same existing amount as in [CM.sub.FB] giving rise to apoptotic effect in HL-60 cancer cells. Rather, we have seen a synergistic property of cordycepin on reduction of cell viability (Fig. 6) and induction of cancer cell apoptosis while in combination with [CM.sub.MY] (Fig- 7). In addition, although the both [CM.sub.MY] and [CM.sub.FB] exhibited anti-cancer effects, it is seemingly HL-60 leukemia cells appear the most sensitive to water extracts of C. militaris (Fig. 2).


Recently many researchers have shown that folk medicinal mushrooms produce anti-viral, anti-cancer, anti-inflammatory and immunomodulatory effects on cultured cells and animal models (Patel and Goyal, 2012). Among which, both C. sinensis and C. militaris provide a great deal of bio-chemical resource with potent biological and pharmacological properties of anti-cancer activities (Patel and Goyal, 2012). Current effort has directed toward identifying the compounds responsible for mediating these biological effects such as polysaccharides and nucleosides appearing as major candidates (Das et al., 2010). With regard to the latter, interestingly our results revealed a synergistic property of cordycepin in the [CM.sub.MY]-mediated cell death as a result of achieving significant apoptosis by reconstituting the level of cordycepin to the same amount in the [CM.sub.FB]. In other words, although cordycepin is a strong inducer of apoptosis at high concentration, at low concentration, however, the presence of additional, unknown factors in [CM.sub.MY] or [CM.sub.FB] are able to augment cordycepin-induced apoptosis or vice versa. Previous findings also show a synergistic effect of cordycepin on causing cell death upon the co-treatment of hydroxyurea (Wehbe-Janek et al., 2007), cisplatin (Chen et al., 2013) or tumor necrosis factor [alpha] (TNF-[alpha]) (Kadomatsu et al., 2012).


To the best of our knowledge, our study on the comparison of [CM.sub.FB] and CMMY-mediated cell death provides the first demonstration that [CM.sub.FB] can effectively lead to greater apoptosis than [CM.sub.MY] by inhibiting the cell growth of leukemia cells by promoting the activation of caspase 3. In our study, the water extract of our cultivated C militaris fruiting bodies had much higher concentrations of cordycepin than the [CM.sub.MY]. Nevertheless, considering the potential roles of cordycepin-mediated cell death in various types of cancer cell lines, our discovery of the synergistic property of cordycepin with [CM.sub.MY] extracts may have an important biological and/or clinical implication for anti-cancer therapies. It is thus reasonable to speculate that cordycepin-mediated cell death might also contribute to the progress of the other cell death inducers. In this regard, the fact that [CM.sub.FB] exhibits excellent potential to induce apoptosis supports such a possibility.


Conflict of interest

Authors report no conflict of interest.


We thank Drs. Shean-Shong Tzean, Jun-Yang Liou, Nai-Chun Lin and Pei-Jen Chen for excellent technical assistance and critical comments. This study was supported by grants from the Mucho Biotech. Inc, Taiwan to T.-L. Shen (101E32004 and 101E32075).


Ahmed, A.F., El-Maraghy, N.N., Abdel Chaney, R.H., Elshazly, S.M., 2012. Therapeutic effect of captopril, pentoxifylline, and cordyceps sinensis in pre-hepatic portal hypertensive rats. Saudi J. Gastroenterol. 18,182-187.

Baik, J.S., Kwon, H.Y., Kim, K.S., Jeong, Y.K., Cho, Y.S., Lee, Y.C., 2012. Cordycepin induces apoptosis in human neuroblastoma SK-N-BE(2)-C and melanoma SKMEL-2 cells. Indian J. Biochem. Biophys. 49, 86-91.

Berenbaum, M.C., 1989. What is synergy? Pharmacol. Rev. 41,93-141.

Buenz, E.J., Bauer, B.A., Osmundson, T.W., Motley, T.J., 2005. The traditional Chinese medicine Cordyceps sinensis and its effects on apoptotic homeostasis. J. Ethnopharmacol. 96,19-29.

Chen, L.S., Stellrecht, C.M., Gandhi, V., 2008. RNA-directed agent, cordycepin, induces cell death in multiple myeloma cells. Br. J. Haematol. 140,682-391.

Chen, Y.H., Wang, J.Y., Pan, B.S., Mu, Y.F., Lai, M.S., So, E.C., Wong, T.S., Huang, B.M., 2013. Cordycepin enhances cisplatin apoptotic effect through caspase/MAPK pathways in human head and neck tumor cells. Onco Targets Ther. 6,983-998.

Cho, H.J., Cho, J.Y., Rhee, M.H., Kim, H.S., Lee, H.S., Park, H.J., 2007. Inhibitory effects of cordycepin (3'-deoxyadenosine), a component of Cordyceps militaris, on human platelet aggregation induced by thapsigargin. J. Microbiol. Biotechnol. 17,1134-1138.

Choi, S., Lim, M.H., Kim, K.M., Jeon. B.H., Song, W.O., Kim, T.W., 2011. Cordycepin-induced apoptosis and autophagy in breast cancer cells are independent of the estrogen receptor. Toxicol. Appl. Pharmacol. 257,165-173.

Das, S.K., Masuda, M., Sakurai, A., Sakakibara, M., 2010. Medicinal uses of the mushroom Cordyceps militaris: current state and prospects. Fitoterapia 81,961-968.

Imesch, P., Hornung, R., Fink, D., Fedier, A., 2011. Cordycepin (3'-deoxyadenosine), an inhibitor of mRNA polyadenylation, suppresses proliferation and activates apoptosis in human epithelial endometriotic cells in vitro. Gynecol. Obstet. Invest. 72,43-49.

Ji, D.B., Ye, J., Li, C.L., Wang, Y.H., Zhao.J., Cai, S.Q., 2009. Antiaging effect of Cordyceps sinensis extract. Phytother. Res. 23,116-122.

Kadomatsu, M., Nakajima, S., Kato, H., Gu, L., Chi, Y., Yao, J., Kitamura, M., 2012. Cordycepin as a sensitizer to tumour necrosis factor (TNF)-alpha-induced apoptosis through eukaryotic translation initiation factor 2alpha (elF2alpha)- and mammalian target of rapamycin complex 1 (mTORC1)-mediated inhibition of nuclear factor (NF)-kappaB. Clin. Exp. Immunol. 168,325-332.

Kan, W.C., Wang, H.Y., Chien, C.C., Li, S.L., Chen, Y.C.. Chang, L.H., Cheng, C.H., Tsai, W.C., Hwang, J.C., Su, S.B., Huang, L.H., Chuu, J.J., 2012. Effects of extract from solid-state fermented Cordyceps sinensis on type 2 diabetes mellitus. Evid. Based Complement. Altern. Med. 2012,743107.

Kim, H., Naura, A.S., Errami, Y., Ju, J., Boulares, A.H., 2011. Cordycepin blocks lung injury-associated inflammation and promotes BRCA1-deficient breast cancer cell killing by effectively inhibiting PARP. Mol. Med. 17, 893-900.

Koc, Y., Urbano, A.G., Sweeney, E.B., McCaffrey, R., 1996. Induction of apoptosis by cordycepin in ADA-inhibited TdT-positive leukemia cells. Leukemia 10, 1019-1024.

Lee, H.J., Burger, P., Vogel, M., Friese, K., Bruning, A., 2012, The nucleoside antagonist cordycepin causes DNA double strand breaks in breast cancer cells. Invest. New Drugs 30,1917-1925.

Lee, S.Y., Debnath, T., Kim, S.K., Lim, B.O., 2013. Anti-cancer effect and apoptosis induction of cordycepin through DR3 pathway in the human colonic cancer ceil HT-29. Food Chem. Toxicol. 60,439-447.

Li, X.T., Li, H.C., Li, C.B., Dou, D.Q., Gao, M.B., 2010. Protective effects on mitochondria and anti-aging activity of polysaccharides from cultivated fruiting bodies of Cordyceps militaris. Am. J. Chin. Med. 38,1093-1106.

Lim, L, Lee, C., Chang, E., 2012. Optimization of solid state culture conditions for the production of adenosine, cordycepin, and D-mannitol in fruiting bodies of medicinal caterpillar fungus Cordyceps militaris (L.:Fr.) Link (Ascomycetes). Int. J, Med. Mushrooms 14,181-187.

Lo, H.C., Hsu, T.H., Tu, S.T., Lin, K.C., 2006. Anti-hyperglycemic activity of natural and fermented Cordyceps sinensis in rats with diabetes induced by nicotinamide and streptozotocin. Am. J. Chin. Med. 34,819-832.

Lo, H.C., Tu, S.T., Lin, K.C., Lin, S.C., 2004, The anti-hyperglycemic activity of the fruiting body of Cordyceps in diabetic rats induced by nicotinamide and streptozotocin. Life Sci. 74, 2897-2908,

Niwa, Y., Matsuura, H., Murakami, M., Sato, J., Hirai, K., Sumi, H., 2013. Evidence that naturopathic therapy including Cordyceps sinensis prolongs survival of patients with hepatocellular carcinoma. Integr. Cancer Ther. 12,50-68.

Patel, S., Goyal, A., 2012. Recent developments in mushrooms as anti-cancer therapeutics: a review. 3 Biotech 2,1-15.

Reis, F.S., Barros, L., Calheiha, R.C., Ciric, A., van Griensven, L.J.. Sokovic, M., Ferreira, I.C., 2013, The methanolic extract of Cordyceps militaris (L.) Link fruiting body shows antioxidant, antibacterial, antifungal and antihuman tumor cell lines properties. Food Chem. Toxicol. 62,91-98.

Shrestha, B., Han, S.K., Lee, W.H., Choi, S.K., Lee, J.O., Sung, J.M., 2005. Distribution and in vitro fruiting of Cordyceps militaris in Korea. Mycobiology 33,178181.

Stone, R., 2008. Mycology. Last stand for the body snatcher of the Himalayas? Science 322,1182.

Stone, R., 2010. Bhutan. Improbable partners aim to bring biotechnology to a Himalayan kingdom. Science 327,940-941.

Tuli, H.S., Sharma, A.K., Sandhu, S.S., Kashyap, D., 2013. Cordycepin: a bioactive metabolite with therapeutic potential. Life Sci. 93, 863-869.

Wagner, H., Ulrich-Merzenich, G., 2009. Synergy research: approaching a new generation of phytopharmaceuticals. Phytomedicine 16,97-110.

Wehbe-Janek, H., Shi, Q., Kearney, C.M., 2007. Cordycepin/Hydroxyurea synergy allows low dosage efficacy of cordycepin in MOLT-4 leukemia cells. Anticancer Res. 27,3143-3146.

Yan, X.F., Zhang, Z.M., Yao, H.Y., Guan, Y., Zhu, J.P., Zhang, LH., Jia, Y.L, Wang, R.W., 2013. Cardiovascular protection and antioxidant activity of the extracts from the mycelia of Cordyceps sinensis act partially via adenosine receptors. Phytother. Res. 27,1597-1604.

Yang, H.Y., Leu, S.F., Wang, Y.K., Wu, C.S., Huang, B.M., 2006. Cordyceps sinensis mycelium induces MA-10 mouse Leydig tumor cell apoptosis by activating the caspase-8 pathway and suppressing the NF-kappaB pathway. Arch. Androl. 52, 103-110.


Article history:

Received 8 April 2014

Received in revised form 10 June 2014

Accepted 27 July 2014

Shang-Min Chou (a,1), Wan-Jung Lai (b,1), Tzu-Wen Hong (b,c), Jui-Ya Lai (b), Sheng-Hong Tsai (b), Yen-Hsun Chen (b), Sz-Hsien Yu (b), Cheng-Hsiang Kao (b), Richard Chu (b), Shih-Torng Ding (d,e), Tsai-Kun Li (a,e,*), Tang-Long Shen (c,e,**)

(a) Department and Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Taipei 100, Taiwan

(b) Mucho Biotechnology Inc., Taipei 106, Taiwan

(c) Department of Plant Pathology and Microbiology, National Taiwan University, Taipei 106, Taiwan

(d) Department of Animal Science and Technology, National Taiwan University, Taipei 106, Taiwan

(e) Center for Biotechnology, National Taiwan University, Taipei 106, Taiwan

* Corresponding author at: Department and Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Taipei 100, Taiwan.

Tel.: +886 2 2312 3456x88287/88294; fax: +886 2 2391 5293.

** Corresponding author at: Department of Plant Pathology and Microbiology, National Taiwan University, Taipei 106, Taiwan. Tel.: +886 2 3366 4602; fax: +886 2 2363 6490.

E-mail addresses: (T.-K. Li), (T.-L Shen).

(1) Both authors provided equal contributions to this work.
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Article Details
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Author:Chou, Shang-Min; Lai, Wan-Jung; Hong, Tzu-Wen; Lai, Jui-Ya; Tsai, Sheng-Hong; Chen, Yen-Hsun; Yu, Sz
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Oct 15, 2014
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