The Inhibitory Effect of Cordycepin on the Proliferation of MCF-7 Breast Cancer Cells, and Its Mechanism: An Investigation Using Network Pharmacology-Based Analysis

Cordyceps militaris is a well-known medicinal mushroom. It is non-toxic and has clinical health benefits including cancer inhibition. However, the anticancer effects of C. militaris cultured in brown rice on breast cancer have not yet been reported. In this study, we simultaneously investigated the anticancer effects of cordycepin and an extract of C. militaris cultured in brown rice on MCF-7 human breast cancer cells using a cell viability assay, cell staining with Hoechst 33342, and an image-based cytometric assay. The C. militaris concentrate exhibited significant MCF-7 cell inhibitory effects, and its IC50 value was 73.48 µg/mL. Cordycepin also exhibited significant MCF-7 cell inhibitory effects, and its IC50 value was 9.58 µM. We applied network pharmacological analysis to predict potential targets and pathways of cordycepin. The gene set enrichment analysis showed that the targets of cordycepin are mainly associated with the hedgehog signaling, apoptosis, p53 signaling, and estrogen signaling pathways. We further verified the predicted targets related to the apoptosis pathway using western blot analysis. The C. militaris concentrate and cordycepin exhibited the ability to induce apoptotic cell death by increasing the cleavage of caspase-7 -8, and -9, increasing the Bcl-2-associated X protein/ B-cell lymphoma 2 (Bax/Bcl-2) protein expression ratio, and decreasing the protein expression of X-linked inhibitor of apoptosis protein (XIAP) in MCF-7 cells. Consequently, the C. militaris concentrate and cordycepin exhibited significant anticancer effects through their ability to induce apoptosis in breast cancer cells.


Introduction
The death rate due to breast cancer is 14%, and this malignancy accounts for 23% of all cancer cases. Therefore, it remains a significant clinical and societal issue worldwide [1]. Patients diagnosed with breast cancer may receive drug therapy, surgery, hormone therapy, and/or radiotherapy. These treatments have a significant and long-term impact on patients' lives owing to the side effects of drug therapy and hormone therapy and the limitations of surgery and radiotherapy [2,3]. Among them, cisplatin is one of the most widely used chemotherapeutic drugs for clinical treatment [4]. Although the use of cisplatin causes toxic adverse effects such as nephrotoxicity, this drug is still rice. Then, it was concentrated under low pressure. For the in vitro and in vivo experiments, it was freeze-dried.

Determination of Cell Viability
The cell viability of MCF-7 cells in C. militaris concentrate and cordycepin was evaluated using an Ez-Cytox Cell Viability Assay Kit (Dail Lab Service Co., Seoul, Korea). Briefly, cells with a density of 1 × 10 4 cells/100 µL were seeded onto 96-well plates. After incubation for 24 h, C. militaris concentrate and cordycepin at various concentrations were added. After treatment for 24 h, 10 µL of Ez-Cytox solution was added and incubated for 30 min. The absorbance was measured at 450 nm (absorbance for live cells) in a microplate reader (PowerWave XS; Bio-Tek Instruments, Winooski, VT, USA). Cisplatin, which is one of the most widely used chemotherapeutic drug for breast cancer, was used as a positive control.

Cell Staining with Hoechst 33342
The nuclear condensation of MCF-7 cells induced by C. militaris concentrate and cordycepin was evaluated using Hoechst 33342 staining (Sigma Aldrich, St. Louis, MO, USA). Briefly, cells at a density of 4 × 10 5 cells/3 mL were seeded onto 6-well plates. After incubation for 24 h, C. militaris concentrate and cordycepin at various concentrations were added. After treatment for 24 h, 2 µL of Hoechst 33342 solution was added, and the plates were then incubated for 10 min. Subsequently, the stained cells were analyzed using an IX50 fluorescent microscope equipped with a charge-coupled device (CCD) camera (Olympus, Tokyo, Japan).

Image-Based Cytometric Assay
The apoptotic death of MCF-7 cells induced by C. militaris concentrate and cordycepin was evaluated using an image-based cytometric assay kit (Invitrogen, Temecula, CA, USA). Briefly, cells at a density of 4 × 10 5 cells/3 mL were seeded onto 6-well plates. After incubation for 24 h, C. militaris concentrate and cordycepin at various concentrations were added. After treatment for 24 h, cells were collected and suspended in Annexin V Binding Buffer (Life Technologies, Carlsbad, CA, USA). Then, Annexin V Alexa Fluor 488 (Invitrogen) was added and the cells were incubated in the dark for 30 min at 20 ± 5 • C. Next, apoptotic cells stained with green Annexin V Alexa Fluor 488 were counted and analyzed with a Tali image-based cytometer (Invitrogen).

Western Blot Analysis
The signaling pathways of MCF-7 cell apoptosis induced by C. militaris concentrate and cordycepin were evaluated using western blot analysis. Briefly, cells at a density of 4 × 10 5 cells/3 mL were seeded onto 6-well plates. After incubation for 24 h, C. militaris concentrate and cordycepin at various concentrations were added. After treatment for 24 h, cells were collected using a cell scraper and lysed using radio immunoprecipitation assay (RIPA) buffer (Cell Signaling Technology, Inc., MA, USA) containing 1× ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride (PMSF). The protein concentrations of samples were determined using a Pierce BCA Protein Assay Kit (Thermo Scientific, Carlsbad, CA, USA). Protein (20 µP) was mixed with 4× NuPAGE LDS Sample Buffer (Thermo Scientific, Carlsbad, CA, USA). After boiling for 10 min, the proteins were separated on precast 4-15% Mini-PROTEAN TGX (Tris-Glycine. eXtended) gel (Bio-Rad, Hercules, CA, USA) and then electrotransferred onto polyvinylidene fluoride (PVDF) transfer membranes. To enhance the detection of proteins, membranes were incubated with specific primary antibodies to Bcl-2-associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), cleaved caspase-7, cleaved caspase-8, X-linked inhibitor of apoptosis protein (XIAP), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, Inc.), followed by incubation with horseradish peroxidase-conjugated secondary goat anti-rabbit antibody (Cell Signaling Technology, Inc.). The bound antibodies were visualized using Pierce ECL (enhanced chemiluminescence) Western Blotting Substrate (Rockford, IL, USA) and a FUSION Solo Chemiluminescence System (PEQLAB Biotechnologie GmbH, Erlangen, Germany).

Network Pharmacological Analysis
Network pharmacological analysis was performed by predicting targets of ingredients in traditional Chinese medicine (TCM) and constructing a compound-target network. The potential targets of cordycepin were obtained from the TCM-mesh (http://mesh.tcm.microbioinformatics.org) based on the combined score [44]. The combined score is calculated by a model that predicts drug-target interaction using random forests [45]. To get as many potential targets of cordycepin as possible, we set the threshold of the combined score to 150 (minimal threshold suggested by TCM-mesh). Then, a compound-target network was constructed by linking herbal ingredients and their predicted targets using the drug-target interaction information.
Gene set enrichment analysis (GSEA) based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database was performed to identify potential pathways related to breast cancer using Enrichr [46]. Enrichr computes enrichment by assessing multiple gene-set libraries (e.g., gene ontology, KEGG, and Online Mendelian Inheritance in Man (OMIM)) and calculates adjusted p-values, Z-scores and combined scores for the gene lists of interest (target genes). The combined score is calculated by the logarithm of multiplication of the p-value and z-score (note that this combined score is different from the combined score in the TCM-mesh).

Statistical Analysis
The data are presented as the mean ± standard deviation (SD). Statistical significance was determined using the Student's t-test and the hypergeometric test. p-Values less than 0.05 were considered statistically significant.

Effects of Cordyceps Militaris Concentrate and Cordycepin on MCF-7 Breast Cancer Cell Viability
Cell viability assays were performed to evaluate the cytotoxic effects of the C. militaris concentrate and cordycepin on MCF-7 cells. Cisplatin was used as the positive control. C. militaris concentrate suppressed cell proliferation in a concentration-dependent manner ( Figure 1A, IC 50 : 73.48 ± 2.76 µg/mL). Cordycepin also significantly suppressed cell proliferation in a concentration-dependent manner ( Figure 1B, IC 50 : 9.58 ± 0.99 µM). Cisplatin suppressed cell proliferation in a concentration-dependent manner, but its IC 50 value was higher than that of cordycepin ( Figure 1C, IC 50 : 48.50 ± 1.84 µM). Based on these results, we investigated the effects of C. militaris concentrate and cordycepin on morphological changes, which contributed to the inhibition of MCF-7 cell viability, using a phase contrast inverted microscope and phase contrast fluorescence microscope ( Figure 1D). After treatment with C. militaris (100 µg/mL) and cordycepin (25, 50 µM), most cells detached from the cell culture plate, and apoptotic cell morphology such as membrane blebbing, cell shrinkage, and cell condensation [47] increased in comparison to the untreated normal cells.

Network Pharmacological Analysis of Cordycepin
Network pharmacological analysis was conducted to elucidate the systems-level mechanism of cordycepin. We constructed and visualized the compound-target network of cordycepin using Cytoscape [48]. In the compound-target network, nodes represented cordycepin and its targets, whereas edges represented the predicted interactions between cordycepin and targets. The targets of cordycepin were colored to indicate the pathways related to breast cancer. The pathways related to breast cancer obtained from literature were as follows: apoptosis pathway, breast tumor kinase pathway, cyclin-dependent kinases pathway, estrogen signaling pathway, hedgehog signaling pathway, human epidermal growth factor receptor 2 (HER2) signaling pathway, mammalian target of rapamycin signaling pathway, notch signaling pathway, phosphoinositide 3-kinases (PI3K)-protein kinase B (Akt) signaling pathway, and Wingless and INT-1 (Wnt)/β-catenin signaling pathway [49,50]. The numbers of related targets for the apoptosis, estrogen signaling, notch signaling, and hedgehog signaling pathways were 5, 2, 2, and 2, respectively ( Figure 2). B-cell lymphoma 2 (BCL2) was simultaneously related to the apoptosis, estrogen signaling, and hedgehog signaling pathways. Next, we conducted GSEA to identify potential pathways that are significantly associated with the targets of cordycepin among the pathways related to breast cancer. The results showed that the apoptosis pathway had the highest overlap and lowest p-value for the targets of cordycepin. In addition, the hedgehog signaling, p53 signaling, and estrogen signaling pathways were significantly associated with the targets of cordycepin (Table 1).

Effects of the Cordyceps Militaris Concentrate and Cordycepin on Apoptosis
To verify the anticancer mechanisms identified through network pharmacological analysis, image-based cytometric assay and western blot analysis were performed for the C. militaris concentrate and cordycepin to evaluate their apoptotic effect on MCF-7 cells. To explore the effects of C. militaris and cordycepin on apoptotic cell death, MCF-7 cells were stained with Annexin V. After treatment with C. militaris concentrate (100 µg/mL) and cordycepin (25, 50 µM), apoptotic cells stained with enhanced Annexin V (green fluorescence) increased as compared to untreated normal cells ( Figure 3A). The percentage of apoptotic cells was significantly increased to 22.50 ± 2.17%, 40.06 ± 3.49%, and 52.63 ± 2.56% after treatment with C. militaris concentrate (100 µg/mL) and cordycepin (25 µM, 50 µM), respectively ( Figure 3B). In keeping with these results, DNA fragmentation identifying apoptotic cells was increased after treatment with C. militaris concentrate (100 µg/mL) and cordycepin (25, 50 µM). Blue fluorescence of Hoechst 33342 on these cells was brighter than that of untreated normal cells ( Figure 4A). To identify the apoptotic mechanism of C. militaris concentrate and cordycepin, western blot analysis was performed. After treatment with C. militaris concentrate (100 µg/mL) and cordycepin (25, 50 µM), the Bax (pro-apoptotic Bcl-2 family)/Bcl-2 (anti-apoptotic Bcl-2 family) ratio was increased by more than two times. And cleavage of caspase-8, which is an apoptotic initiator, was significantly increased by more than five times. Furthermore, cleavage of caspase-7, an apoptotic effector caspase, was significantly increased by more than three times. Meanwhile, treatment of cordycepin inhibited XIAP, which is the X-linked inhibitor of apoptosis protein ( Figure 4B).

Discussions
In our investigations on the Cordyceps militaris concentrate and cordycepin to examine in vitro anticancer activity on MCF-7 human breast cancer cells, we observed that both the C. militaris concentrate and cordycepin exhibited a significant cytotoxic effect on MCF-7 cells. The IC 50 value of the latter was about five times lower than that of cisplatin, which is currently the most effective platinum-based drug for breast cancer [51]. The extract of C. militaris exhibited cytotoxic effects on the 4T1 murine breast cancer cell line in a recent study, and the extract (100, 200, and 400 µg/mL) inhibited cell viability in a concentration-dependent manner [52]. In addition, cordycepin (100 µM) has been reported to inhibit cell viability below 50% in MCF-7, MDA-MB 231, and MDA-MB-435 human breast cancer cells [53]. These results indicate that cordycepin exhibits cytotoxic effects on various types of breast cancer cell lines.
In the present study, the C. militaris concentrate and breast cancer cell line also suppressed cell proliferation of the MDA-MB-231 breast cancer cell line in a concentration-dependent manner ( Figure S1). Although active concentrations on MDA-MB-231 were higher than on MCF-7, C. militaris concentrate and cordycepin also inhibited cell proliferation of the MDA-MB-231 breast cancer cell line. Meanwhile, 100 µg/mL of C. militaris concentrate did not affect the viabilities of LLC-PK1 pig kidney epithelial cells and human umbilical vein endothelial (HUVEC) cells, which are normal cell lines ( Figure S2). Cordycepin also did not affect the viabilities of LLC-PK1 and HUVEC cells until 50 µM. We suggest that they are good candidates for targeting breast cancer.
To investigate the detailed cytotoxic effects, a phase contrast inverted microscope was used to evaluate the pattern of cell death in MCF-7 cells. Results of this observation indicated that apoptotic cell morphologies including membrane blebbing, cell shrinkage, and cell condensation were increased after treatment with C. militaris concentrate and cordycepin. These findings were consistent with the results of a previous study, which reported that cell debris and irregularly shaped cells were increased in MCF-7 cells after treatment with cordycepin (100 µM) [54]. However, these are hallmarks of apoptosis but not exact evidence [47]. Therefore, to evaluate apoptotic cell death under treatment with C. militaris concentrate and cordycepin in MCF-7 cells, Hoechst 33342 staining and image-based cytometric assays were used to distinguish apoptotic cells from non-apoptotic cells. Observation with a phase-contrast fluorescence microscope indicated that blue fluorescence from Hoechst 33342-staining of DNA as well as DNA fragmentation, which is a key characteristic of apoptosis [55], were also increased. In keeping with this result, apoptotic cells stained with enhanced Annexin V (green fluorescence) were increased after treatment with C. militaris concentrate and cordycepin.
To elucidate the systems-level mechanism, a compound-target network was constructed based on the predicted targets of cordycepin. The predicted targets were associated with the apoptosis, estrogen signaling, hedgehog signaling, and notch signaling pathways. In addition, GSEA was used to identify pathways that were significantly enriched in the target list of cordycepin. Among the pathways associated with breast cancer, the apoptosis pathway showed the greatest overlap, lowest p-value, and highest combined score.
To further investigate the changes in protein expression involving the apoptotic pathways, western blot analysis was used. The apoptotic pathways involved in the anticancer effects of cordycepin have been extensively studied in various cancers over a long period of time (about 60 years) [36]. Collectively, treatment with cordycepin increases the phosphorylation of terminal deoxynucleotidyl transferase (TdT) by protein kinase A (PKA) [56]. Treatment with cordycepin also induces apoptosis through the mitochondria-mediated intrinsic apoptotic pathways indicated by translocation of Bax from the cytosol to the mitochondria, release of cytochrome c from the mitochondria to the cytosol, and activation of caspase-9. Caspase-9 is activated by apaf-1 (apoptosome) and activates caspase-3 and -7 [57,58]. In addition, treatment with cordycepin induces death receptor-mediated extrinsic apoptotic pathways, as indicated by the activation of caspase-8, which stimulates two parallel cascades. One is the activation of caspase-3 and -7 and the other is the release of cytochrome c induced by the truncated Bid (pro-apoptotic Bcl-2 family), which translocates to the mitochondria and results in an increase in the Bax/Bcl-2 ratio [59,60]. Furthermore, treatment of cordycepin inhibited XIAP. XIAP is bound to caspase-3, -7, and -9 and suppresses cell death by caspase overproduction [61].
However, the MCF-7 cells used in our study were caspase-3 deficient [62,63]. Therefore, caspase-7 was confirmed as the apoptotic effector caspase. In the MCF-7 cells, C. militaris concentrate and cordycepin induced the cleavage of caspase-8, which are apoptotic initiator caspases. This resulted in the cleavage of caspase-7, an apoptotic effector caspase, and an increase in the Bax/Bcl-2 ratio. These results suggest that the C. militaris concentrate and cordycepin induced apoptosis via both mitochondrial-mediated intrinsic, and death receptor-mediated extrinsic apoptotic pathways. The targets of cordycepin related to the apoptosis pathway were summarized using KEGG Mapper ( Figure 5). . Apoptosis pathways (hsa04210) and targets of cordycepin. The pathway maps were constructed using KEGG mapper. The orange colored box represents a predicted target to interact cordycepin, the uncolored box represents a target that is expected to not interact, the gray colored box represents a target which is outside the predictable target list in TCM-mesh, and the purple-bordered box represents a validated target by western blot.
Among the targets of cordycepin investigated by western blot analysis, three target genes were also found in the predicted target lists (CASP8, Bax, Bcl-2 and XIAP). The accordance rate was significant (p-value < 0.001, hypergeometric test), which supports the reliability of the predicted results. Of the two mismatched targets, CASP9 was predicted not to interact with cordycepin (incorrect prediction), and CASP7 was not found in the predicted target list of TCM-mesh (unpredictable target).
In summary, we performed several in vitro experimental methods including cell viability assays, cell staining with Hoechst 33342, image-based cytometric assays, and western blot analysis to evaluate the apoptotic effects of the C. militaris concentrate and cordycepin on MCF-7 human breast cancer cells. The C. militaris concentrate and cordycepin exhibited potent cytotoxic effects on MCF-7 cells and could increase the cleavage of caspase-7 -8, and -9, and increase the Bax/Bcl-2 protein expression ratio in MCF-7 cells. Additionally, we applied network pharmacological analysis to predict potential targets and pathways of cordycepin and the targets related to the apoptosis pathway were validated in vitro.

Conclusions
In this study, the C. militaris concentrate cultured in brown rice and cordycepin isolated from C. militaris induced the cell death of MCF-7 human breast cancer cells. Network pharmacological analysis revealed a systems-level mechanism of cordycepin that could explain its suppressive effect on the proliferation of breast cancer cells, and the targets associated with the apoptosis pathway were verified by further experiments. This study provides a promising potential application not only for C. militaris cultured in brown rice itself but also for cordycepin isolated from C. militaris as a traditional medicine and alternative to chemotherapy in breast cancer. It also suggests that the strategy of integration of network pharmacology and experimental validation is a powerful tool to obtain a deep understanding of the mechanisms of natural products that act on multiple targets.

Conflicts of Interest:
The authors declare no conflict of interest.