1. Introduction {#s0005}
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The number of people suffering from cancer in the world will continue to grow steadily, according to the latest report about the trend of global cancer released by the World Health Organization (WHO). It is estimated that the number of newly-increased cancer patients will reach 19 million or even more by 2025. Cancer is the second killer of human death in the world ([@b0005]). Cancer is caused by an imbalance between the progression of cell cycle and programmed cell death (Apoptosis) ([@b0135]). Therefore, the majority of anticancer medical drugs exert their anti-proliferative activity through cell cycle arrest and induction of apoptosis ([@b0010], [@b0055]). The cytotoxic nucleoside analogues were the first chemotherapeutic agents used for the therapy of cancer. The some researched cytotoxic nucleoside analogues are isolated from *Cordyceps militaris* ([@b0195]).

Cordycepin was first found from the fermented broth of the medicinal mushroom *Cordyceps militaris*, which is the fungus that grows parasitically on lepidopteron larvae and insect pupae ([@b0050]). The genus *Cordyceps* is well-known in traditional Chinese medicine and exhibits a variety of clinical health effects including immunomodulatory, anticancer, antioxidant, anti-inflammatory and anti-microbial activities ([@b0200], [@b0285]). Recently, more and more studies have demonstrated Cordycepin, as one bioactive compound of *Cordyceps militaris*, have abroad roles of anti-tumor ([@b0075], [@b0085], [@b0220], [@b0225], [@b0290]). However, little is known about the active ingredients as well as the mechanism underlying these roles. The review summarizes the anti-tumor mechanism of Cordycepin.

2. Main active components of *Cordyceps militaris* {#s0010}
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2.1. Chemical features of cordycepin {#s0015}
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The structure of Cordycepin is very much similar with cellular nucleoside, adenosine and acts like a nucleoside analogue ([@b0120]). The structure of cordycepin comprises a purine (adenine) nucleoside molecule attached to one ribose sugar moiety. The chemical synthesis of cordycepin is completed mainly through the replacement of the OH group at the 3′-position in the ribofuranosyl moiety with H, generating a deoxy analogue of adenosine ([Fig. 1](#f0005){ref-type="fig"}) ([@b0205]).Fig. 1The difference of chemical structures between Cordycepin and adenosine.

2.2. Function of cordycepin {#s0020}
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Cordycepin has many biological and pharmacological actions in immunological, hepatic, renal, cardiovascular systems as well as an anti-cancer agent. Those functions are related to its structure ([@b0200]). During the process of RNA synthesis (transcription), some enzymes are not able to distinguish between an adenosine and Cordycepin which leads to incorporation of Cordycepin to induce premature termination of transcription ([@b0040], [@b0070]). In addition, The IC50 (the concentration at which 50% inhibition of cell growth was achieved) of cordycepin in human gallbladder cancer cell lines NOZ and GBC-SD cells at 48 h was approximately 19.2 μg/mL and 398.1 μg/mL, respectively ([@b0240]). For human lung cancer cell lines. IC50 of cordycepin was 60 μg/ml ([@b0090]). The function of Cordycepin treatment in tumor is dependent on tumor types and concentration ([@b0045], [@b0065]).

The high dosage of Cordycepin can block mTOR (mammalian target of rapamycin) signaling pathway ([@b0255]). The name mTOR has been derived from the drug rapamycin, because the drug inhibits mTOR activity. Some of mTOR inhibitors have been tested as anti-cancer drugs, since they suppress cancer through mTOR signaling pathway ([@b0020], [@b0170]). The study found that Cordycepin can activate AMPK which blocks the activity of mTORC1/mTORC2 complex. The inactivated complex cannot activate AKT 1 kinase fully, which suppress mTOR signal transduction inhibiting translation, and further cell proliferation and growth ([@b0255]). Those functions of Cordycepin elucidate their structure--function relationship, and further explain the anti-tumor roles of the compound. Cordycepin had been shown to regulate AMPK/mTORC1 signaling pathway to down-regulate multiple drug resistant to HIF-1α in GBC-SD gallbladder cancer cells ([@b0260]). The anti-tumor roles and mechanisms of Cordycepin are descripted detail below.

3. Cordycepin inhibits tumor growth {#s0025}
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Many complementary and alternative medicine are developed in applications of cancer prevention and therapy due to chemotherapy resistance and metastasis ([@b0250]). Traditional Chinese medicine is one treatment for complementary and alternative therapy ([@b0245], [@b0270]). Cordycepin is an active compound and has been used in cancer treatment in past studies.

3.1. Induction of tumor apoptosis {#s0030}
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Cordycepin can induce cancer cell apoptosis in caspase-dependent pathways. Apoptosis of Human liver cancer (HepG2) cells were induced by the activation of caspase, interaction between Fas and FADD, and modulation of the protein levels of Bid and tBid ([@b0180]). Cordycepin also decreased human bladder carcinoma cells (T24 cells) survival, which was regulated by the activation of A3 adenosine receptor and the subsequent inactivation of Akt pathways, leading to the increases in cleaved Caspase-3 and apoptosis ([@b0025]). In addition, Cordycepin reduced cell viability, inhibited cell proliferation, and enhanced lactate dehydrogenase release and reactive oxygen species (ROS) accumulation of human breast cancer cell (MCF-7 and MDA-MB-231 cells) through up-regulating the activation of pro-apoptotic proteins, such as caspase-3, 8, 9 and suppressing the expression of the anti-apoptotic protein, B-cell lymphoma 2 (Bcl-2) ([@b0235]).

Cordycepin induced the apoptosis of human renal cancer cells by triggering the MKK7-JNK signaling pathway through inhibition of anti-apoptotic protein cellular caspase 8 (FLICE)-like inhibitory protein (c-FLIP) expression and the consequent activation of the Bax/caspase-3/PARP-mediated pathway ([@b0085]). In human Non-Small Cell Lung Cancer (NSCLC), Cordycepin-induced apoptosis was also associated with down-regulation of protein c-FLIP, which inhibited the activity of caspase-8. Cordycepin inhibited cell growth by inducing apoptosis and autophagy. The cordycepin-stimulated autophagy were mediated by suppressing mTOR signaling pathway in lung cancer cells. In addition, suppression of autophagy could also elevate the protein level of c-FLIP which indicated cordycepin-triggered autophagy promoted the degradation of c-FLIP. Therefore, Cordycepin induced apoptosis through autophagy-mediated downregulation of c-FLIP in human NSCLC cells. In addition, cordycepin also inhibits the ERK/Slug signaling pathway through the activation of GSK3β which, upregulates Bax to result in apoptosis of lung cancer cells ([@b0090]). Taken together, cordycepin may serve as one promising therapeutic compound, which acts on multiple molecular targets in lung cancer treatment ([@b0280]).

Cordycepin also induces cancer cell apoptosis in caspase-independent pathways. Cordycepin decreased cell mitosis and EGFR signaling in one murine oral tumor mouse model. In accordance, the treatment distinctly reduced the levels of ki-67 and EGFR signaling molecules to induce cancer cell apoptosis ([@b0075]). For human lung adenocarcinoma, Cordycepin induced cancer cell apoptosis by caveolin-1-upregulated JNK/Foxo3a signaling pathway, and significantly decreased tumor volume in nude mice ([@b0100]). Cordycepin also increase ROS levels and induce apoptosis in MA-10 mouse Leydig tumor cells but not cause cell death of primary mouse Leydig cells on moderate concentration through down-regulating the p38 MAPK and PI3K/AKT signaling pathways ([@b0155]) ([Table 1](#t0005){ref-type="table"}).Table 1Inhibition roles of Cordycepin on tumor growth.Tumor types (cell lines)Mechanism of anti-tumorMolecular targetsReferencesHuman gastric cancer (SGC-7901)Induction of apoptosisPI3K/AKT↑([@b0145])Human non-small cell lung cancer cellsInduction of apoptosis/autophagyc-FLIPL↓([@b0280])Human liver cancer (HepG2)Induction of apoptosisCaspase-8, Fas, FADD↑([@b0180])Human renal cancer cells (TK-10)Induction of apoptosisMKK7, JNK↑([@b0085])Human uterine cervical cancer cell (ME180 and HeLa cell)Induction of G2/M arrestCyclin A2↓([@b0175])Human Leukemia cells (NB-4 and U937 cells)Induction of apoptosis/cell cycle arrestCyclin A2, cyclin E, and CDK2↓ p53↑([@b0130])Human Bladder cancer (T-24)Induction of apoptosisA3 adenosine receptors↑([@b0025])Human bladder cancer cell (5637 and T-24 cells)G2/M cell cycle arrestPhosphorylation of c-Jun([@b0115])Human breast cancer cells (MCF-7 and MDA-MB-231)Induction of apoptosisCaspase-3,8,9↑, BCL-2↓([@b0235])Murine oral cancer (4NAOC-1)Induction of apoptosis, decrease cell mitosis and EGFR signalingCaspase-3↑ EGFR, IL-17RA↓([@b0075])Murine Leydig tumor cell (MA-10)Induction of apoptosisp38 MAPK↑([@b0075])

3.2. Cell cycle arrest {#s0035}
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Cordycepin incorporates mitochondrial-mediated apoptosis in gastric cancer cell (SGC‑7901 cells) with regulating mitochondrial extrinsic pathways by inhibition of A~3~ adenosine receptor (A~3~AR) and drive activation of death receptor DR3, which promote the activation of PI3K/Akt protein expression as well as collapse of mitochondrial membrane potential (MMP). Phosphorylation of PI3K/Akt and DNA damage by cordycepin induced the production of ROS and regulated SGC‑7901 cell cycle cessation at S phase ([@b0145]). Cordycepin also increased radio-sensitivity in human uterine cervical cancer cells, such as ME180 and HeLa cells, and induced the increased number of those tumor cells in the G2/M phase, which is related to the induction of p53-mediated apoptosis and modulation of the expression of cell cycle checkpoint molecules ([@b0175]). The increased expression of p53 by Cordycepin treatment promoted the release of cytochrome c from mitochondria to the cytosol, to further activate caspase-9 and promote the apoptosis of leukemia cells (NB-4 and U937 cells) ([@b0130]). In addition, cordycepin inhibits the expression of cyclin A2, cyclin E, and CDK2, which leads to the accumulation of those leukemia cells in S-phase through the activation of Chk2-Cdc25A pathway ([@b0130]).

Recently, Lee at al found that Cordycepin causes p21WAF1-mediated G2/M cell cycle arrest by upregulating c-Jun N-terminal kinase activation in human bladder cancer cells. They blocked JNK function using JNK-specific inhibitor and small interfering RNA of JNK to rescue cordycepin-dependent p21WAF1 expression and decrease of cell cycle proteins ([@b0115]). These results suggest that cordycepin could be an effective treatment for bladder cancer.

3.3. Resistance of cancer stem cell {#s0040}
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Cancer stem cells (CSCs) are a limitless cell source for the initiation and maintenance of cancer cells. CSCs can generate cancer cells through the stem cell processes of self-renewal and differentiation into multiple tumor cell types ([@b0015], [@b0210]). Thus, The intrinsic resistance of CSCs to conventional therapy is regarded as a potential therapeutic target of cancer ([@b0160]). Activation of the Wnt/β-catenin pathway is required for the survival and development of CSCs, such as leukemia stem cells (LSCs) ([@b0150]). Therefore, targeting β-catenin is considered a therapeutic strategy for the treatment of leukemia. cordycepin can block the effect of β-catenin in leukemia cells by regulating GSK-3β to inhibit the growth of LSCs ([@b0105]). CSCs escape chemotherapy and lead to chemo-resistance due to the induction of TGF-β. Cordycepin efficiently inhibited cell viability, the percentage of ovarian cancer stem cells, and the levels of matrix metalloproteinases (MMPs) in TGF-beta-induced SKOV-3 ovarian cancer cells. Thus, cordycepin acted as a complementary agent for ovarian cancer therapy that against chemoresistance ([@b0230]).

4. Regulation of cordycepin on tumor microenvironment {#s0045}
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4.1. Inhibition of migration and invasion of tumor cell {#s0050}
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Cordycepin inhibited the migration and invasion of human oral squamous cell carcinoma (OSCC) cell through upregulating E-cadherin and downregulating N-cadherin protein expression, implying the inhibition of Cordycepin on epithelial-mesenchymal transition (EMT) ([@b0280]). In addition, Cordycepin have been shown to suppress the migration of the human glioblastoma cell lines U87MG and LN229 in transwell and wound healing assays *in vitro*, since Cordycepin decreased protein expression of integrin α1, focal adhesion kinase (FAK), p-FAK, paxillin and p-paxillin. The lysosomal inhibitor NH4Cl can block the ability of cordycepin to inhibit focal adhesion protein expression and glioma cell migration. The protein phosphatase inhibitors Calyculin A and okadaic acid also blocked the cordycepin-mediated reduction in p-Akt, p-FAK and further suppress tumor cell line migration. Hematoxylin and eosin staining of mouse xenografts demonstrated that brain tumor sizes were reduced after Cordycepin treatment *in* vivo. Thus, cordycepin inhibited the migration and invasion of human glioblastoma cells by affecting lysosomal degradation and protein phosphatase activation ([@b0080]). These data are in consistent with the finding that cordycepin inhibits the migration and invasion of LNCaP cells (human prostate carcinoma cells). Cordycepin significantly downregulated the activity of tight junctions and suppressed the expression and activity of MMP-2 and MMP-9, which regulated tumor metastasis. These anti-metastatic roles were mediated by inactivation of the phosphoinositide 3-kinase (PI3K)/Akt pathway in LNCaP cells ([@b0095]).

4.2. Blockage of tumor metastasis {#s0055}
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Anti-metastatic activities of cordycepin were demonstrated in mouse models where cordycepin inhibited B16 mouse melanoma liver metastasis *in vivo* ([@b0110]). The potential roles of cordycepin in melanoma cell metastasis and the underlying molecular mechanisms were addressed further. Zhang et al. found that cordycepin could suppress melanoma invasion via MMPs and metastasis via actomyosin machinery through LXR/RXR activation-dependent upregulation of miR-33b. Cordycepin also suppressed the expressions of HMGA2, Twist1 and ZEB1 through miR-33b. The up-regulation of miR-33b by cordycepin inhibited melanoma metastasis *in vivo* ([@b0295]). In another *in vivo* mouse melanoma studies, Yoshikawa at al demonstrated that adenosine-5′-diphosphate (ADP)-induced platelet aggregations accelerated lung metastasis on mouse melanoma. Cordycepin treatment reduced the number of metastatic lung nodules through blocking ADP-induced platelet aggregations ([@b0275]). Those data indicated that cordycepin inhibit melanoma metastasis through different anti-metastatic mechanisms.

Cordycepin can suppress mitochondrial fusion-induced EMT in ovarian carcinoma cells through inhibiting estrogen-related receptor (ERR)-α, which is a co-transcription factor for gene expressions associated with mitochondrial fusion Thus, cordycepin suppresses metastasis and migration of ovarian carcinoma cells via inhibiting mitochondrial activity ([@b0225]). In addition, Cordycepin also blocked EMT through regulating TGF-β ([@b0230]).

Solid tumors grow fast if they induce the development of new blood vessels, a process known as tumor angiogenesis, which is the main process of tumor growth and metastasis ([@b0030], [@b0035]). Angiogenesis was assessed using a tube formation assay ([@b0265]). Anti-angiogenic drugs have been broadly used for clinical studies to suppress the growth and metastasis of tumors ([@b0060]). Cordycepin inhibited tube formation (total length of tubular structure) of human umbilical vein endothelial cell line (HUVEC) and the migration of those cells. Cordycepin also efficiently suppressed the invasion and migration of hepatocellular carcinoma cell line (HepG2) ([@b0140]) ([Table 2](#t0010){ref-type="table"}).Table 2Inhibition roles of Cordycepin on tumor migration and metastasis.Tumor types (cell lines)Anti-metastatic mechanismMolecular targetsReferencesHuman oral squamous cell carcinomaInhibition of epithelial-mesenchymal transition (EMT)E-cadherin, N-cadherin↓([@b0190])Human hepatocellular carcinoma (HepG2)Anti-metastatic and anti-angiogenic([@b0140])Human glioblastoma cell (U87MG and LN229)Inhibition of tumor cell motilityLysosomal degradation, protein phosphatase activation↑([@b0080])Human prostate carcinoma (LNCaP)Inhibition of migration and invasion of tumorAKT↓([@b0095])Human ovarian carcinoma (OVCAR-3)EMTMitochondrial activity↓, Estrogen-related receptor α↓([@b0225])Human melanomaInhibition of invasion and metastasismiR-33b↓, HMGA2, Twist1, ZEB1↑([@b0295])

4.3. Disruption between cancer cells and mesenchymal stromal cells (MSCs) {#s0060}
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Mesenchymal stromal cells (MSCs), as the main cell type of tumor microenvironment, promote tumor growth and metastasis, and stromal cells support tumor progression and resistance to chemotherapy ([@b0165], [@b0215]). Thus, targeting the niche-based microenvironment may be one new approach for cancer therapy ([@b0185]). Cordycepin reduces the numbers of CD34+CD38-cells in leukemia such as U937 and K562, and induces Dkk1 expression to disrupt the association of both leukemia and MSCs. Cordycepin also suppressed cell attachment of leukemia with MSCs and downregulates N-cadherin in leukemia and VCAM-1 in MSCs ([@b0125]). Therefore, the results indicated the potential of cordycepin as a multitarget drug in anti-metastatic therapy.

5. Conclusion and future prospect {#s0065}
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Numerous studies have shown that Cordycepin as one valuable compound, can inhibit many malignant tumors through different pathways. Since Cordycepin-induced death of cancer cells are performed via multi-target pathways, it is difficult to some extent for cancer cells to develop drug resistance. Moreover, another advantage of Cordycepin is that the small side effect is shown when inhibiting the growth and progression of cancer cells. Therefore, Cordycepin may be considered as one wonderful drug candidate for cancer treatment.

This work was supported by Jilin province science and technology development program (20160201001YY); Jilin province science and technology development program (20170520043JH); Foundation of Jilin Educational Committee (JJKH20170726KJ); the Intramural Research Program at the NCI, NIH; the National Natural Science Foundation of China (Grant No. 81572868); Natural Science Foundation of Shangdong (Grant No. ZR2014ZM023).

Peer review under responsibility of King Saud University.
