Cordycepin may affect tumour cells through a variety of mechanisms, including promoting tumour cell apoptosis, inhibiting tumour cell proliferation, invasion and metastasis, and regulating the expression of tumour-related genes. Additionally, in animal models, cordycepin also inhibited tumour growth (28). Fig. 1 shows a diagram of the antitumour mechanism of cordycepin.

The MAPK/ERK pathway plays an important role in cancer development. This pathway is involved in biological processes such as cell proliferation, survival, differentiation and metastasis (32). Abnormal MAPK/ERK pathway activation is closely associated with the development and progression of various cancers (33), such as lung cancer, stomach cancer, ovarian cancer (34–36). Therefore, this pathway has become an important target for cancer treatment. Currently, several inhibitors of the MAPK/ERK pathway have been developed and used to treat several cancers (37). These inhibitors can reduce the proliferation and metastasis of cancer cells by inhibiting the MAPK/ERK pathway, thereby achieving therapeutic effects (38). In a study on oral cancer, it showed that the small molecule inhibitor OTX008 induced the MAPK signaling pathway early (39).

The study by Zhou et al (40) explored the anticancer potential and mechanism of cordycepin in nasopharyngeal carcinoma. RNA sequencing (RNA-seq) combined with in vitro experiments revealed that the expression levels of ERK1/2, phosphorylated ERK1/2 and β-catenin were significantly decreased after cordycepin treatment. This suggests that cordycepin may be a novel drug candidate for nasopharyngeal cancer treatment. In addition, in oesophageal cancer, cordycepin induces chromatin condensation, activates the caspase cascade, induces apoptotic signalling, regulates Bcl-2 family members to increase cell apoptosis and alters cyclin-dependent kinases 1 and 2. The expression of cyclin B1 led to G₂/M phase arrest (41). A mechanistic study revealed that inactivation of the ERK pathway was involved in the antitumour effect of cordycepin and the same result was also observed in vivo (41). Moreover, cordycepin was confirmed to activate caspase-3, caspase-9 and cytochrome C to induce apoptosis, activate the checkpoint kinase 2 pathway and downregulate cyclin A2 and cyclin-dependent kinase 2 (CDK2) phosphorylation-related genes, S-phase arrest and DNA damage (42). Furthermore, the same study showed that cordycepin blocks the MAPK pathway by inhibiting the expression of Ras and the phosphorylation of ERK, and the blockade of the Ras/ERK pathway with fibroblast growth factor receptor 2 effectively inhibited the growth of pancreatic cancer cells (42).

Under normal conditions, the phosphatidylinositol 3-kinase (PI3K/AKT/mTOR) signalling pathway regulates cell proliferation and survival through a series of signal transduction and phosphorylation cascades (43). As an initiation factor of the pathway, PI3K is activated under the signal stimulation of molecules, including growth factors and cytokines, to catalyse the conversion of phosphatidylinositol diphosphate into phosphatidylinositol triphosphate (PIP3) (44). PIP3 activates AKT, which affects downstream activity, including that of FOXO transcription factors and mTOR proteins, to regulate cell proliferation and activity (45). In ovarian, stomach and breast cancer, abnormal activation of the PI3K/AKT/mTOR signalling pathway may be due to PIK3CA mutations such as deletion of the phosphatase and tensin homolog gene, overexpression of AKT and an increase in mTOR activity (46–48). Abnormally activated PI3K/AKT/mTOR signalling promotes the abnormal proliferation, survival and metastasis of cancer cells (49).

In a concentration-dependent manner, treatment of bladder cancer cells with cordycepin significantly reduced the survival rate of the cells and activated exogenous and endogenous cell apoptosis pathways, increased the caspase effect and resulted in polymerase cleavage (50). By increasing the ratio of Bax/Bcl-2, disrupting the integrity of mitochondria and promoting the release of cytochrome C, mechanistic study showed that cordycepin induces cell apoptosis by activating exogenous and endogenous apoptotic pathways and ROS-dependent inactivation of the PI3K/AKT signalling pathway in human bladder cancer T24 cells (50). In another study, cluster analysis of mRNA expression profiling and western blot analysis was performed on cordycepin-treated testicular cancer cells. The results showed that cordycepin regulates the FOXO/P15/P27, PERK-eIF2α and IRE1-XBP1UPR pathways. This study also found that activation of the AKT and MAPK pathways may lead to cancer cell resistance to cordycepin (51). In addition, cordycepin has a strong hypolipidemic effect. Therefore, it has been found that cordycepin-mediated endoplasmic reticulum oxidoreductase 1α/mTOR/SRE binding transcription factor 1 axis inhibits lipid metabolism and transfer in CCA cells in vivo and in vitro. After treating CCA cells with simvastatin and exogenous cholesterol, the same results were obtained, further confirming that cordycepin inhibits CCA migration through the regulation of lipid metabolism (52). These data indicate that cordycepin could be used as a novel drug for the clinical treatment of CCA and may improve the prognosis of patients with CCA.

In cells, the AMP-activated protein kinase (AMPK) signalling pathway inhibits the growth and proliferation of cancer cells while inducing apoptosis (53). It also inhibits the synthesis and secretion of vascular endothelial growth factor (VEGF) to prevent angiogenesis, thereby inhibiting tumour growth and metastasis (54). In addition, activation of the AMPK pathway can also inhibit autophagy in cancer cells, thereby reducing the growth rate of tumour cells (55). In terms of metabolism, activation of the AMPK pathway can promote the metabolism of glucose and fat, thereby reducing blood glucose and blood lipid levels while increasing intracellular ATP production (56). The AMPK pathway has been confirmed to be associated with the development of cancer (57). Since tumour cells usually depend on glycolysis to generate energy, activation of the AMPK pathway is considered to interfere with the metabolism of cancer cells (58). In conclusion, the AMPK signalling pathway plays an important role in cancer.

In a study on non-small cell lung cancer (NSCLC) cells, compared with cisplatin alone, cordycepin significantly inhibited the effect of cisplatin on cell proliferation and the promotion of cell apoptosis. Moreover, reversing cisplatin resistance in NSCLC through the AMPK and AKT signalling pathways could lead to the development of a potential treatment strategy for overcoming cisplatin resistance in patients with NSCLC (59). In another NSCLC study, cells with EGFR mutations were more sensitive to cordycepin treatment than the control cells (60). In ovarian cancer, cordycepin was found to activate AMPK signalling to transduce downstream target proteins to induce autophagy-dependent cell death (61). Based on the aforementioned results, the present authors considered that cordycepin alone or in combination with currently available targeted therapies might be an additional option for the treatment of lung cancer, especially for patients with EGFR-mutant lung cancer.

The Hh pathway is composed mainly of the Hh ligand, the intracellular protein patched (Ptch) and the signal transduction protein GLI family zinc finger (GLI). In the inactive state, Hh ligands bind to the Ptch protein on the cell surface. When Hh signalling is activated, the Hh ligand is released to dissociate from the Ptch protein (62,63). Persistent activation of the Hh signalling pathway is associated with the development of a variety of tumours, such as skin, brain and pancreatic cancer (64). Therefore, studying the Hh pathway is highly important for understanding the underlying mechanisms of cancers and identifying new therapeutic targets.

For the first time, Liu et al (65) studied the function of the Hh pathway in the effect of cordycepin on human breast cancer cells and the results showed that cell apoptosis induced by cordycepin led to an increase in the p53-upregulated modulator of apoptosis, Cytochrome C, Fas cell surface death receptor, death receptors 4/5 and caspase-3 and also led to the inhibition of Bcl-2, X-linked inhibitor of apoptosis protein and PDGFR-α. It also inhibits the expression of the Hh pathway and the transcriptional activity of GLI (65). The blockage of cordycepin-mediated cell apoptosis, epithelial-mesenchymal transition (EMT) and the Notch pathway after GIL knockout plays an important role in the effect of cordycepin on breast cancer. Further in vivo studies revealed that cordycepin reduced the volume and weight of xenograft tumours, affected proliferation, apoptosis and EMT, and affected the expression of matrix metalloproteinase-related proteins in cancer cells but had no side effects (65). Analysis of RNA-seq data showed that the Hh pathway was the most enriched in breast cancer tissues, and by analysing Hh pathway markers and assessing changes in expression in xenografts, the Hh pathway was shown to play an important role in the anti-breast cancer effect of cordycepin (66).

The Wnt signalling pathway completes signal transduction through the extracellular Wnt protein and intracellular receptors, ligands and transcription regulators (67). The specific cascades involved include the Wnt/β-catenin, the canonical Wnt signalling, the Wnt-planar cell polarity and the Wnt-Ca²⁺ signalling pathways (68). In tumours, abnormal activation of the Wnt signalling pathway is common, as activation of the Wnt signalling pathway maintains stem cell properties and promotes tumour development (69). Therefore, the Wnt signalling pathway is an important target in tumour treatment.

Wnt/β-catenin signalling is required for the development and maintenance of leukaemia stem cells (LSCs) in acute myeloid leukaemia (AML) (70). Cordycepin downregulates the expression of the Wnt target genes MYC and prominin-1 (key factors for the maintenance of stem cells) through Wnt inhibitory factor 1 and Dickkopf-1 (Dkk1). These results provide new insights into the involvement of cordycepin-mediated molecular circuits in pharmacological inhibition and enhanced targeting (71). Wnt/β-catenin and its regulatory complex in AML treated with cordycepin can potentially target LSCs by reducing cell viability and stimulating LSC apoptosis, which supports the potential use of cordycepin as an adjuvant in the treatment of AML (70). Therefore, cordycepin may be effective in the treatment of AML.

Cordycepin inhibits the growth of colorectal cancer (CRC) cells in vitro and may accelerate cancer cell apoptosis by inducing Bax translocation to the mitochondrial membrane (72). The same study also found that cordycepin-induced Bax translocation-induced cell apoptosis and reintroduction of Bax expression could restore the ability of cells to induce apoptosis. These results indicate that cordycepin may be a new agent for the treatment of Bax-deficient cancers (72). Cordycepin significantly inhibited the proliferation of tongue cancer cells in a dose-dependent manner and further induced the upregulation of Bax, caspase-3, caspase-9 and caspase-12 at the mRNA and protein levels while downregulating the increase in the level of the antiapoptotic gene Bcl-2. In addition, cordycepin effectively inhibited the growth of tongue cancer tumours in a mouse xenograft model (73). For the first time, Fong et al (74) confirmed the cytotoxic effect of cordycepin on endometrial cancer cells. The authors observed cell cycle arrest at the G₀/G₁ phase in cells treated with different concentrations of cordycepin, cisplatin or a combination of the two agents. In addition, the antitumour effect of the combination treatment was improved compared with that of a single compound, with fewer adverse drug reactions.

A previous study showed that C-Myc is a downstream target of cordycepin and is positively associated with cell cycle pathways. In retinoblastoma (RB), cordycepin was found to inhibit the expression of cyclin D1 and overexpression of c-Myc could reverse this effect, thereby inhibiting the malignant biological behaviour of RB cells (75). In leukaemia, after hyperthermia and cordycepin combined treatment, the MAPK pathway significantly increased cell apoptosis, the level of matrix metalloproteinases also significantly decreased and ROS generation increased. Combination treatment also downregulates the expression of cyclin-dependent kinase 1 and cyclin B1 proteins and induces cell cycle arrest in the G₂/M phase (76). Cordycepin treatment inhibited the growth of cervical cancer cells, increased apoptosis and interfered with the cell cycle, especially through prolongation of the S phase. After cordycepin treatment, the mRNA levels of the cell cyclins CDK2, Cyclin-A2 and Cyclin-E1 were downregulated, but the expression of apoptosis-related proteins did not significantly change. The aforementioned results indicate that cordycepin is effective at treating cervical cancer cells (77). Microarray analysis of cordycepin-treated MA-10 mouse stromal tumour cells revealed that cordycepin downregulated the expression levels of FGF9, FGF18, FGFR2 and FGFR3 in MA-10 cells. Moreover, the pathway prediction results showed that the inhibition of ERK1/2 and retinoblastoma protein/E2F transcription factor 1 pathways, the cell cycle pathway and the expression of the FGFR1-4 protein might inhibit the growth of FGF9-induced testicular tumours (78). In a mouse allograft model, the amount of FGF9-induced tumour growth in the cordycepin treatment group was significantly lower than that in the PBS treatment group (78). In summary, cordycepin could be used as a new anticancer drug for tumours and play a role in cancer treatment.

Autophagy is an important biochemical process in cells. Its main function is to maintain cell homeostasis and vitality by decomposing and recycling damaged proteins, organelles and other cellular components (79). Autophagy mainly includes microautophagy, macroautophagy and chaperone-mediated autophagy, and among these processes, macroautophagy is the most extensively studied and understood (80). Autophagy has dual roles in tumours. In the early stage, autophagy may suppress tumour formation; after tumour progression to a certain stage, autophagy may switch to a role in promoting tumour survival and progression (81). Therefore, in tumour treatment, the regulatory and intervention strategies for autophagy need to be precisely adjusted according to the specific situation to achieve the best therapeutic effect.

In oral squamous cell carcinoma cells, the cordycepin-mediated inhibition of cancer cell migration and invasion was also altered when autophagy was inhibited by chloroquine, indicating that the migration and invasion inhibited by cordycepin may be mediated by autophagy (82). After cordycepin treatment, autophagy was induced, the fluorescence intensity of monodansylcadaverine fluorescence intensity (MDC)- and MDC-positive cells increased, and the expression level of the LC3 gene increased. Mechanistic study have shown that high doses of cordycepin inhibit cell death and invasion by inducing stress, while low doses inhibit invasion through autophagy-dependent focal adhesion kinase (FAK)/Akt/MMP2 and MMP9 pathways (82). Cordycepin can increase the level of Dkk1 and inhibit β-catenin signalling in human ovarian cancer cells. These results indicate that cordycepin may promote the cleavage of caspase-3 and inhibit the growth of ovarian cancer cells by coordinating autophagy and Dkk1/β-catenin signalling. Taken together, these data indicate that cordycepin may inhibit the growth of ovarian cancer cells through coordinated autophagy and Dkk1/β-catenin signalling (83). Cordycepin activates DNA-dependent protein kinase and ERK and triggers centrosome expansion to inhibit cell proliferation and destroy the cytoskeleton but does not affect the growth of normal placental cells (84). In addition, cordycepin treatment activated autophagy, and the inhibition of cordycepin-induced cell death by chloroquine prevented cordycepin-induced cell death, revealing the potential therapeutic effect of cordycepin.

Cordycepin inhibited the growth and migration of and promoted the apoptosis of CRC cells in a dose-dependent manner, increased the infiltration of CD4⁺ T cells, CD8⁺ T cells, M1 macrophages and NK cells in the tumour immune microenvironment and inhibited the expression of trisodium phosphate in tumour cells (85). Reducing the binding of thrombospondin to CD47 allows more T cells to infiltrate tumours and inhibits the growth of CRC in a mouse tumour model (31). These results indicate that the adjuvant of cordycepin with other antitumour drugs can further enhance antitumour immunity and inhibit the growth of CRC and is a potential adjuvant drug for the immunotherapy of CRC. For the first time, Panwong et al (86) reported the ability of cordycepin to sensitize NK cells to toxicity in CCA cells. Compared with cordycepin or NK treatment alone, cordycepin treatment significantly increased the expression of the tumour necrosis factor-related apoptosis-inducing ligand receptor (TRAILR) in KKU-4A cells. The increased expression of the TRAILR may help cordycepin regulate immune activity. However, how cordycepin promotes NK cell activation remains unclear. These results indicate that cordycepin can be further developed as a substitute immunomodulatory drug in adoptive NK cell therapy.

In a previous study, combination treatment with cordycepin and an anti-CD47 antibody significantly inhibited tumour growth and prolonged the survival of tumour-bearing mice. The flow cytometry results showed that when cordycepin was used in combination with the cytotoxic T-lymphocyte antigen 4 (CTLA-4) blocker, the proportion of M1 and M2 macrophages was decreased, the presence of tumour-infiltrating CD8⁺ T cells was significantly increased, and the proportion of M2 macrophages was decreased (27). Reducing the number of Foxp3 Tregs in the tumour microenvironment significantly inhibited tumour growth and prolonged the survival of tumour-bearing mice (27). In summary, the results of this study indicate that the combination of cordycepin and a CTLA-4 blockade can change the effector and exhaustion states of CD8⁺ T cells, thereby enhancing CD8⁺ T cell-mediated antitumour immunity in the tumor microenvironment (TME). According to the results of another single-cell RNA-seq study, the combination of cordycepin and a CD47 antibody could reactivate macrophages, reverse polarization, increase the proportion of M1 macrophages, decrease the proportion of M2 macrophages, regulate the proportion of CD8⁺ T cells, and prolong the progression-free survival of patients with malignant tumours (87). In summary, these results prove that the use of cordycepin may enhance the function of immune cells, which is valuable for the treatment of various types of cancer.

A study on triple-negative breast cancer showed that cordycepin can inhibit the expression of twist-related protein 1 and snail homolog 2 to inhibit the EMT signalling pathway. IT was also reported that combination therapy with cordycepin and thymoquinone had synergistic effects on the inhibition of tumour metastasis (88). Cordycepin significantly decreased FAK expression and induced p53 and p21 expression in endothelial cell (EC) to impair angiogenesis and tumour growth and reduce hepatocellular carcinoma (HCC) tumour growth in a xenograft model. The present authors further examined the number of ECs in the tumour area in tumour-bearing mice. However, due to severe necrosis in the tumour area, the number of ECs or blood vessels could not be quantified (89). In another HCC study, cordycepin significantly inhibited IκBα phosphorylation, limited the nuclear translocation of P65 and activated tissue transcription factors, resulting in the downregulation of CXCR4 expression and the inhibition of migration and invasion in HCC cells. JSH-23 (an NF-κB pathway inhibitor) can inhibit the migration of liver cancer cells and was found to exert a synergistic effect when combined with cordycepin (90). These findings indicate that cordycepin can be used as a potential adjuvant in cancer treatment and may prevent HCC metastasis when it is used in combination with other therapeutic compounds.

Cordycepin activates the AMPK and MAPK signalling pathways, inhibits lipid metabolism and EMT processes, and significantly inhibits the proliferation, colony formation and migration of gastric cancer cells (91). Cancer cell proliferation is inhibited by downregulating MYC mRNA and protein expression and upregulating miR-2a in CRC cells. Furthermore, MYC overexpression inhibited the expression of miR-26a, while miR-26a expression was restored after cordycepin treatment. These results indicated that the inhibition of CRC cell proliferation by cordycepin might be mediated by the MYC/miR-26a pathway (92).

Cordycepin can slow the migration, growth and clonality of uveal melanoma and other invasive malignant tumours with low adenosine deaminase (ADA) levels, indicating that ADA may be a predictive biomarker of the cordycepin response and that tumours that are resistant to monotherapy may be sensitive to combination therapy. The present authors also found that cordycepin inhibited heat shock protein 90, thereby destroying its function and leading to the degradation of client proteins such as hypoxia-inducible factor, protein kinase B, extracellular signal-regulated kinase and EGFR, resulting in the activation of proteins in oncogenic signalling pathways, and reported a new mechanism of action of cordycepin (93). However, only in vivo studies were performed and the effect of cordycepin on the growth of orthotopic xenografts in other types of tumours needs to be evaluated.

Based on a combination of theoretical and experimental studies, the design and preparation of cordycepin-encapsulating liposomes were performed. Molecular dynamics simulations and free energy calculations showed that the phosphatidylcholine lipid environment was conducive to the adsorption of cordycepin. Cordycepin passively permeates into the PC lipid bilayer without causing damage to the membrane and tightly binds to the polar group of lipids by flipping its deoxyribose sugar toward the centre of the bilayer. In an in vitro study of colon cancer cell lines, cordycepin-encapsulated liposomes enhanced the anticancer activity of cordycepin (94). The study indicated that cordycepin-encapsulated liposomes may be effective drug candidates for the treatment of CRC.

Radiation therapy uses radiation energy to damage the DNA of cancer cells, prevent their ability to proliferate and divide, and shrink or even eliminate tumours, and can be used to treat breast cancer, lung cancer, lymphoma, head and neck cancer and other tumor (95–98). A study showed that in mouse stromal tumour cells, the combination of cordycepin and radiotherapy has synergistic effects on the inhibition of cancer cell viability by activating exogenous and endogenous caspase pathways, cell cycle arrest and endoplasmic reticulum stress. Moreover, in animal experiments, it also reduced the tumour mass of stromal tumours (99). Subsequently, after combination treatment, ROS accumulated, heme oxygenase-1 protein levels decreased in mouse mesenchymal tumour cells and DNA damage-related signalling pathways were activated, including the ataxia-telangiectasia mutation induced by double-strand and single-strand breaks/checkpoint kinase 2 (Chk2), ataxia-telangiectasia mutations and Rad3 related (ATR)/Chk1 signaling axes were identified. The tumour volume, size and weight were reduced, and high expression of γ-H2AX was observed in in vivo tumour tissues after combination therapy (100). Network pharmacology analysis revealed the systemic mechanism underlying the inhibitory effect of cordycepin on the proliferation of breast cancer cells. Several studies showed that after radiation exposure, breast cancer cells cultured with cordycepin increased the levels of intracellular ROS and γ-H2AX lesions. However, the expression levels of nuclear factor erythrocyte 2-related factors and a series of downstream genes were downregulated after cordycepin treatment, sensitizing breast cancer cells to radiation through the Nrf2/HO-1/ROS axis. In addition, cordycepin promoted G₂/M arrest and apoptosis in breast cancer cells, thereby inhibiting cell proliferation in vitro and in vivo after radiation. Therefore, these study results indicate that cordycepin may be used as a radiosensitizer during clinical breast cancer radiotherapy (101,102). Another study showed that cordycepin can repair DNA damage in oral squamous cell carcinoma cells through Chk1 phosphorylation and prolong radiotherapy-induced G₂/M phase arrest. According to in vivo experiments, the growth inhibitory effect of the combination of cordycepin and radiotherapy on xenografts was greater than that of radiotherapy alone, and no excessive toxicity was observed (103). However, the clinical application of cordycepin in enhancing radiosensitivity needs further verification.

Treatment with cordycepin or cisplatin alone failed to induce cell death in bladder cancer cells, but these two drugs in combination induced mitochondrial membrane depolarization, decreased the expression of antiapoptotic proteins and increased the expression of proapoptotic proteins. A high expression level of multidrug resistance protein 1 (MDR1) in bladder cancer results in cisplatin resistance. After cordycepin treatment, the inhibition of MDR1 promoter activity reduces MDR1 expression and induces resensitization of cancer cells to cisplatin (104). The combination of cordycepin and cisplatin enhances the sensitivity of osteosarcoma cells to cisplatin, increases apoptosis and inhibits the growth and invasion of osteosarcoma cells by activating AMPK and inhibiting the AKT/mTOR signalling pathway (105). RNA-seq analysis revealed 72 genes whose expression was significantly different from that of the other genes and in which different signalling pathways may be regulated by cordycepin; in addition, the expression levels of ERK1/2, phosphorylated ERK1/2 and β-catenin were significantly downregulated (40). A relevant study confirmed that cordycepin enhances the chemosensitivity of oesophageal cancer cells to cisplatin through the activation of AMPK and the inhibition of the AKT signalling pathway, and in vivo and in vitro experiments demonstrated the synergistic effects of these two drugs (106). In clinical practice, combination therapy comprising cordycepin and cisplatin may be a potential treatment method for improving the treatment of oesophageal cancer (106). In addition, sensitization to other chemotherapeutic drugs was also found when other chemotherapeutic drugs were used in combination. Cordycepin combined with temozolomide inhibits EMT and regulates the proliferation, migration and apoptosis of glioblastoma cells (107). Cordycepin combined with doxorubicin regulates the EMT of tumour cells to inhibit cell invasion and migration, and this synergistic effect was proven through network pharmacology and in vitro experiments (108). Cordycepin and apatinib inhibited the VEGF/PI3K/AKT pathway, reduced cell proliferation, inhibited cell migration and invasion, altered the cell cycle to increase cell apoptosis, and exerted synergistic anticancer effects (109).

Therefore, cordycepin may be a novel drug candidate for cancer treatment and a candidate for combination therapy with other drugs, but the clinical feasibility of combination treatment still needs to be comprehensively evaluated in future studies, which could provide potential insights for anticancer drug design targets.