Cinobufagin: a promising therapeutic agent for cancer

Abstract

Objectives

Cinobufagin is a natural active ingredient isolated from the traditional Chinese medicine Venenum Bufonis (Chinese: Chansu), which is the dried secretion of the postauricular gland or skin gland of the Bufo gargarizans Cantor or Bufo melanostictus Schneider. There is increasing evidence indicating that cinobufagin plays an important role in the treatment of cancer. This article is to review and discuss the antitumor pharmacological effects and mechanisms of cinobufagin, along with a description of its toxicity and pharmacokinetics.

Methods

The public databases including PubMed, China National Knowledge Infrastructure and Elsevier were referenced, and ‘cinobufagin’, ‘Chansu’, ‘Venenum Bufonis’, ‘anticancer’, ‘cancer’, ‘carcinoma’, and ‘apoptosis’ were used as keywords to summarize the comprehensive research and applications of cinobufagin published up to date.

Key findings

Cinobufagin can induce tumour cell apoptosis and cycle arrest, inhibit tumour cell proliferation, migration, invasion and autophagy, reduce angiogenesis and reverse tumour cell multidrug resistance, through triggering DNA damage and activating the mitochondrial pathway and the death receptor pathway.

Conclusions

Cinobufagin has the potential to be further developed as a new drug against cancer.

Introduction

Cancer is the second leading cause of death in the world.^[1] Around 19.3 million new cases of malignant tumours and 10 million deaths were detected worldwide in 2020, which means that 1/5 of the world’s population will suffer from cancer in their lifetime.^[2] Modern medicine treats cancer mainly by surgery, radiotherapy and chemotherapy, but single surgery is still limited due to postoperative recurrence and cancer cell metastasis, and so on.^{[3, 4]} Presently, the effects of cancer treatment are not very ideal and there is a long way to go to achieve ideal therapy.

China has a long history of using Chinese medicine to treat cancer, with records of malignant tumours dating back 3500 years to the Yin and Zhou dynasties.^[5] Based on the theory of traditional Chinese medicine, tumours are considered to be lumps in the abdomen causing distension and pain. Therefore, herbal medicines are often used to treat cancer by softening hard knots, activating blood circulation and removing blood stasis, and clearing heat and detoxification, such as Curcuma longa L.,^[6]  Epimedium brevicornu Maxim.,^[7]  Astragalus membranaceus (Fisch.) Bunge,^[8]  Panax ginseng C. A. Meyer,^[9]  Salvia miltiorrhiza Bge. ^[10] andVenenum Bufonis^[11]. In addition to inhibiting tumour growth, Chinese medicine can also regulate immunity, enhance the effects of radiotherapy and chemotherapy, as well as reduce the side effects of radiotherapy and chemotherapy, and improve the quality of survival, taking advantage of the whole process of cancer rather than the terminal stage.^[12] Therefore, Chinese medicine is of great importance in the treatment of malignant tumours.

Venenum Bufonis, also known as Chansu and Toad venom, is the dried secretion of the postauricular gland or skin gland of the Bufo gargarizans Cantor or Bufo melanostictus Schneider displaying functions of detoxification, pain relief and enlightenment, and has been used for hundreds of years to treat various diseases in China,^{[13, 14]} although it is now commonly used for anaesthesia, pain relief, anticancer, etc.^{[15, 16]} Chansu is a mixture that contains up to 96 types of bufadienolide monomers and 23 types of indole alkaloids, such as bufalin, cinobufagin, resibufogenin, telocinobufagin and arenobufagin.^{[17, 18]} Cinobufagin is one of the main active ingredients, whose structure is shown in Figure 1. Numerous studies have displayed that cinobufagin has good anticancer activity.^[19–21] It is widely used in the treatment of various malignant tumours such as liver cancer, gastric cancer, non-small cell lung cancer, colorectal cancer, rectal cancer, breast cancer, prostate cancer, cervical cancer, osteosarcoma, melanoma, nasopharyngeal cancer, oral squamous cell carcinoma, and glioblastoma. Its pharmacological effects include induction of tumour cell apoptosis and cell cycle arrest, inhibition of tumour cell proliferation, migration and invasion, reduction of cancer angiogenesis and autophagy, reversal of tumour cell multidrug resistance, triggering DNA damage and relief of cancer pain. This review describes in detail cinobufagin and its antitumour and other biomedical effects and mechanisms, and discusses its toxic effects and pharmacokinetics to explore its potential for development as a tumour suppressor, providing new ideas and insights into the current research of such compounds as shown in Figure 2.

Anticancer Effects

Cancer development is associated with anomalous proliferation of genetically damaged cells.^[22] Drug treatments such as cinobufagin can alter the abnormal activity of the cells through specific mechanisms. Accumulating evidence has revealed the antitumour effects of cinobufagin and herein we summarize the reported mechanisms and presented them more intuitively as outlined in Figure 3. and Table 1.

Induction of tumour cell apoptosis

Apoptosis is a process of programmed cell death characterized by chromatin condensation, DNA fragmentation and the shedding of small fragments in cells,^{[61, 62]} which is beneficial for maintaining the stability of the body’s internal environment. However, apoptosis does not occur naturally due to mutation, hence inducing apoptosis in tumour cells becomes one of the important strategies for the treatment of many cancers.

Cinobufagin can induce apoptosis in tumour cells by activating caspases through the death receptor pathway and the mitochondrial apoptosis pathway. The one of cell surface death receptor (Fas)-mediated extrinsic pathway plays a key role in the apoptosis of hepatocellular carcinoma HepG₂ cells^[48] and prostate cancer DU145 and PC3 cells.^[57] Cinobufagin promotes the upregulation of Fas, which then binds to specific death ligands to activate the caspase cascade reaction to induce apoptosis. In osteosarcoma U2OS and 143B cells,^{[63, 31]} uveal melanoma OCM1 cells,^[41] melanoma A375 cells,^[40] prostate cancer LNCaP, DU145 and PC3 cells,^[64] hepatocellular carcinoma HepG₂ cells^[65] and glioblastoma U87 cells,^[66] cinobufagin exerts pro-apoptotic effect through activation of the mitochondrial pathway. The mitochondria-dependent endogenous apoptotic pathway is dependent on reactive oxygen species (ROS) generation and loss of ΔΨm, resulting in the release of cytochrome C from mitochondria into the cytoplasm, where it binds to Apaf-1 and further activates caspase-9, followed by downstream caspase-3 activation by cleaved caspase-9, resulting in the cleavage of multiple key cellular substrates, including PARP, which in turn induces apoptosis.

The ratio of Bcl-2-associated X (Bax) to Bcl-2 is critical for maintaining drug-induced apoptosis in the mitochondria-mediated apoptosis pathway. In one study mice were intraperitoneally injected with cinobufagin at a dose of 5 mg/kg d⁻¹. After 30 consecutive days, the mRNA expressions of Bcl-2-asociated death promoter (Bad), Bax, caspase-3 and PARP increased, while B-cell lymphoma-2 (Bcl-2) and B-cell lymphoma-xl (Bcl-xl) decreased in uveal melanoma tissues, indicating that cinobufagin can promote apoptosis of cancer cells by regulating Bax/Bcl-2 ratio.^[41] Notch signalling pathway also plays an essential role in the apoptosis of cancer cells.^[67] For example, cinobufagin promotes apoptosis in cholangiocarcinoma QBC939 and RBE cells by inhibiting the Notch pathway.^[42] Similar effects were observed in osteosarcoma U2OS, 143B and MG-63 cells.^[33] In addition, p53 is also involved in the apoptosis of HepG₂ cells.^[68] p73 is a member of the p53 family, which acts as an equivalent to p53 and may be a potential therapeutic target for cancer cells lacking functional p53.^[69] Cinobufagin activates apoptotic signalling pathway by upregulating p73 expression in oesophageal squamous carcinoma EC-109, Kyse-150 and Kyse-520 cells^[44] and hepatocellular carcinoma Huh-7 cells.^[46] In addition to p73, cinobufagin can also act by inhibiting phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT),^[66] glycogen synthase kinase-3β (GSK-3β)/NF-κB^{[49, 34]} and MAPKs signalling pathways.^[56] Besides, cinobufagin can activate the tumour suppressor gene forkhead box O1 (FOXO1)^{[35, 54]}and inhibit c-Myc-related genes^[53] to promote tumour cells apoptosis. By inhibiting the β-catenin signalling pathway, cinobufagin degrades PML-RARA cells and induces apoptosis of NB4 and NB4-R1 cells in a caspase-dependent manner, suggesting a new therapeutic strategy for the treatment of acute promyelocytic leukemia which is a specific malignant haematologic neoplasm, but its exact mechanism needs to be further explored.^[70]

Inhibition of cancer cell proliferation

One of the most important characteristics of cancer cells is that they can proliferate indefinitely and become ‘immortal cells’. Therefore, induction of cancer cell inactivation or inhibition of their proliferation is one of the ways of cancer treatment.

In different cancers, cinobufagin inhibits the activity of cancer cells and its antiproliferation targets are also different. Signal transducer and activator of transcription 3 (STAT3) is associated with promoting tumour cell proliferation and therefore has been used as a therapeutic target to inhibit tumour cell proliferation in many studies.^[71] Cinobufagin inhibits the growth of colorectal cancer cells HCT116, RKO and SW480 by inhibiting STAT3 signalling pathway.^[23] Epidermal growth factor receptor (EGFR) amplification and mutations are present in different types of cancers. In a tumour-bearing nude mouse model inoculated subcutaneously with U87-EGFR cells, intraperitoneal injections of 1 or 5 mg/kg d⁻¹of cinobufagin significantly slowed tumour growth, which is related to inactivation of the EGFR and STAT3 signalling pathways. Cinobufagin also exhibits specific antiproliferative effects in EGFR-expressing specific cancer cells, including glioblastoma, lung cancer, colorectal cancer and hepatocellular carcinoma.^[43]

Besides, Anoctamin1 (ANO1), a calcium-activated chloride channel is also a potential therapeutic target.^[72] Cinobufagin can downregulate the high amplification and expression of ANO1 in the OSCC cell line CAL-27, thereby inhibiting tumour cell viability.^[30] mTORC1 inactivation may also be important for the antiproliferative activity of cinobufagin in CRC cells.^[51] It is well known that activation of RAS-RAF-MEK-ERK, PI3K-AKT and canonical Wnt/β-catenin signalling pathways can promote cancer cell growth. Cinobufagin inhibits hepatocellular carcinoma cell proliferation by attenuating extracellular regulated kinase (ERK) phosphorylation and downregulating c-Myc expression.^[24] As a potent inhibitor of Wnt/β-catenin signalling, cinobufagin reduces lymphoid enhancer-binding factor 1 (LEF1) expression, thereby inhibiting melanoma growth.^[39]

Suppression of invasion and metastasis

Abnormal differentiation, invasion and metastasis are typical biological characteristics of tumour cells.^[73] In general, metastasis of cancer cells makes treatment more difficult and is a key factor in clinical death of oncology patients. Therefore, it is urgent to find ways to treat cancer cells that can inhibit metastasis. Epithelial mesenchymal transition (EMT) is the process by which epithelial cells with malignant phenotype are transformed into mesenchymal stem cells to promote metastasis.^[74] Cinobufagin not only inhibits EMT to suppress the growth, invasion and metastasis of colorectal cancer cells but also affects the expression of EMT protein levels. It induces Enkurin (ENKUR) to inhibit β-linked protein/c-Jun/MYH9 signalling, thereby reducing UBE3A-mediated p53 ubiquitin degradation. As a result, inactivation of EMT signalling leads to metastasis inhibition in nasopharyngeal carcinoma.^[38] Besides, cortactin is also an important factor involved in cancer cell progression and invasion.^[75] Cinobufagin inhibits the expression of cortactin in colon cancer to suppress colon cancer cell invasion and metastasis in vitro and in vivo.^[28] Na/K-ATPase is an integral heterodimeric plasma membrane protein that maintains cell membrane potential and osmotic homeostasis between intracellular and extracellular pathways.^[76] Due to its regulation by several hormones, studies have shown its involvement in abnormal protein expression and in the progression of several cancers.^[77] Bufadienolide is an inhibitor of Na/K-ATPase, is considered a promising source of Na/K-ATPase inhibitory anticancer prototype drugs.^[78] Cinobufagin acts as one of the important compounds of bufadienolide, the other being bufalin. Recent studies suggest that they may have other targets of action. Porcine aminopeptidase N (pAPN) and porcine aminopeptidase A (pAPA), members of the M1 and M17 families, are upregulated on the surface expression of many cells and are positively correlated with cell invasion ability. The results of preliminary screening experiments suggest that cinobufagin non-competitively inhibits pAPN and competitively inhibits pAPA activity, acting as a dual inhibitor. At the cellular level, it was shown that cinobufagin promotes cell death in melanoma MeWo cells and increases DNA degradation of the cells by 35%. Although the link between cell death induction and pAPN is limited, it merits further exploration and provides new ideas for the treatment of cancers associated with pAPN upregulation.^[55]

Cell cycle arrest

The cell cycle is the basis of cell proliferation and blocking the cell cycle also facilitates the antitumour efficacy of drugs. Cinobufagin significantly decreases the expression of CDK2 (cyclin-dependent kinases 2) and cyclin E, which are the regulatory proteins associated with S-phase in HK-1 cells of nasopharyngeal carcinoma, suggesting that cinobufagin blocked the cell cycle in S-phase.^[37]

In melanoma A375 cells, cinobufagin inhibits the expression of cyclin-dependent kinase 1 (CDK1) and cyclin B, and upregulates the levels of ATM serine/threonine kinase, Chk2 (checkpoint kinase 2), p-CDC25C (phosphorylated cell division cycle 25C), thereby inducing G2/M cell cycle arrest.^[40] In addition, Cinobufagin also induces a variety of other cancer cells G2/M-phase arrest, such as HeLa cervical cancer cells, SK-HEP-1 and HepG₂ liver cancer cells and U2OS, MG-63 and SaOS-2 colon cancer cells.^{[34, 45, 60]} However, in MCF-7 breast cancer cells, cinobufagin induces cell cycle arrest in G0/G1 phase.^[58] Cinobufagin-induced cell cycle arrest is unrestricted to specific stages and varies in a variety of non-small cell lung cancer cell lines, suggesting that cinobufagin has different targets in different cells.^[79]

Depression of angiogenesis

New regeneration of blood vessels is often a sign of tumour cell invasion and metastasis.^[80] The generation of tumour blood vessels provides the nutrients needed for tumour growth. When the mass is small, tumour cells can obtain nutrients directly from the surrounding tissue. However, when the mass is larger than 2 mm, the growth of tumour metastasis depends on its own formation of neovascularization. Therefore, inhibition of tumour angiogenesis is one of the effective strategies for current tumour treatment. In a mouse model transplanted with colorectal cancer (CRC) SW480 cells in situ, Li et al. found that cinobufagin inhibited CRC angiogenesis by downregulating activation of the AKT/mTORC1/HIF-1α pathway and triggering MOMP-mediated apoptosis, thus achieving depression of tumour growth.^[25]

Reduction of autophagy

Cellular autophagy can exert bidirectionally modulatory effects on tumour growth. On the one hand, autophagy induces the degradation of anti-apoptotic factors when tumour cells are under the environment of chemotherapy or lack of oxygen and nutrients, promoting cancer progression. But on the other hand, autophagy can stabilize the cell state, stop normal cells on the way to cancer in time, and inhibit tumour neovascularization, reducing tumour growth. Targeting autophagic process will undoubtedly become a new hot spot in antitumour therapy. Cinobufagin induces cellular autophagy by accumulation of GFP- light chain 3 (LC3) fluorescent particles and upregulation of LC3-II levels and the inhibition of autophagy reduces apoptosis of cells. Cinobufagin triggers apoptosis and autophagic cell death in human osteosarcoma U2OS cells through activation of the ROS/JNK/p38 axis.^[36] It can also decrease autophagy to increase apoptosis of gastric cancer cell line SGC-7901, thus exerting the anticancer effects.^[52]

Reversal of drug resistance

Cross-resistance of tumour cells to multiple antitumour drugs with different chemical structures and mechanisms of action is a common clinical phenomenon. It is one of the current research areas to investigate the mechanism of tumour multidrug resistance and discover new effective chemotherapy sensitizers. It is currently believed that the mechanism of multidrug resistance in tumour cells is closely related to the overexpression of ABC transporters, and P-glycoprotein (P-gp) is the most studied ABC transporter.^[81] Cinobufagin can reverse P-gp-mediated multidrug resistance by non-competitively inhibiting P-gp ATPase activity and P-gp efflux function without affecting its expression, indicating that it is a safe and effective P-gp reversal agent.^[27]

Others

Sub-lethal amounts of cinobufagin elicit ROS overload, leading to intense DNA damage and oxidative stress, which in turn induces apoptosis.^[26] Cinobufagin causes DNA damage of oncogenes by reducing the expression of AURKA, mTOR, p-mTOR, p-ERK, eukaryotic translation initiation factor 4E and p-eIF4E.^[47] In addition, cinobufagin has been shown to inhibit growth and trigger DNA damage in human hepatocellular carcinoma cells through proteasome-dependent degradation of thymidylate synthase.^[82] Pain is one of the most common complications of malignant tumours and alleviating cancer pain is beneficial to improve patients’ quality of life. In a paw cancer pain model of mice, cinobufagin significantly increases the pain threshold to alleviate cancer pain mainly through upregulating the expression levels of β-END and μ-OR in hind paw tumours and adjacent tissues.^[83] The analgesic effect of cinobufagin via β-END in bone cancer has also been confirmed.^[84] In addition, M4mAChR is also involved in regulating the pain relief of cinobufagin in bone cancer pain rat model.^[85] Cinobufagin-induced local analgesia may be associated with increased POMC/β-END/μ-OR pathway activity released by invaded CD3/4/8 lymphocytes in cancer tissues.^[86]

As described above, cinobufagin has strong anticancer effects, but it also has other bioactive effects that have potential for further development. Acute lung injury (ALI) due to bacterial infection is still a serious clinical condition with high morbidity and mortality. Studies have shown that in a lipopolysaccharide-induced ALI model, cinobufagin treatment can effectively reverse lung tissue damage such as disrupted alveolar structure, thickened septa, infiltrative oedema and inflammatory cells in the alveoli and interstitium. This reversal was found to be caused by the activation of cellular autophagy in vivo due to reduced levels of inflammatory cytokines such as IL-1β, IL-6 and TNF-α. In addition to this, significant activation of the p53/mTOR pathway, a classical autophagy-associated pathway in vivo, was also observed for cinobufagin.^[87] However, in another study, exposure of dendritic cells to cinobufagin led to the release of IL-1β, recruitment of inflammatory cells to the site of infection and induction of production of the antimicrobial peptides hBD-2 and hBD-3, which have been shown to help fight bacterial infections.^[88] Transforming growth factor beta (TGF-β) is a major promoter of myofibroblast activation and plays a very important role in EMT. Emerging evidence suggests that cinobufagin inhibits the TGF-β1/Smad3 signalling pathway in a concentration-dependent manner, primarily inhibiting fibroblast differentiation without significant toxic effects. More importantly, in vivo experiments have found that cinobufagin ameliorates bleomycin-induced lung fibrosis in mice and reduces the number of inflammatory cells and inflammatory cytokines.^[89] Cinobufagin can also reduce the expression of c-Myc gene to inhibit the growth of leukaemia cells and thus fight against acute myeloid leukaemia.^[90] There is also a study describing the efficacy of cinobufagin as an adjuvant for inactivated Salmonella typhimurium vaccine, stimulating IFN-γ production to enhance Th1 immune responses in vivo.^[91] Then for hepatitis B virus, cinobufagin also exhibited a slight inhibitory effect.^[92]

Toxicity

Cinobufagin belongs to bufadienolide and is one of the active ingredients of Venenum Bufonis. Although cinobufagin has good antitumour efficacy, its toxicity and side effects should also address. High dose cinobufagin is toxic to the heart as it triggers cardiac arrhythmias and to the central nervous system by triggering seizures, muscle spasms or fatal paralysis.^{[93, 94]} Cinobufagin induces acute local ischaemia to cause heart failure, with half maximal inhibitory concentration (IC₅₀)=10⁻⁸ M.^[95] It also induces ROS-related cytotoxicity in human astrocyte cells and promotes the development of brain-related diseases. Cinobufagin at the dose of around 20 nM and above may be toxic to GHA and HCN-2 cells, although it does not induce Ca²⁺ signalling or ROS responses in HCN-2 cells. It may interfere with Ca²⁺ signalling or ROS downstream sensitivity.^[96] In an acute toxicity test, cinobufagin caused the death of Kunming mice (20 ± 2g) after 10–60 min of administration, with an LD₅₀ of 4.78 mg/kg, and the poisoning symptoms include closed eyes, curled up, respiratory depression and reduced activity.^[97] However, in the experiment conducted by our research group, the C57BL/6 mice showed no obvious abnormal symptoms after daily intragastric administration of 20 mg/kg, suggesting that there exists the heterogeneity of the effect of cinobufagin and further exploration is needed.

Pharmacokinetics

Pharmacokinetics is one of the most important reasons affecting the development and marketing of anticancer drugs. Because of the toxic effects of cinobufagin, it is necessary to study its pharmacokinetics and metabolism. The pharmacokinetics of cinobufagin was first described in 1987 by Toma et al.^[98] After intravenous administration of 0.25 mg/kg of cinobufagin to rats, the pharmacokinetic parameters were obtained by HPLC quantitative analysis: elimination half-life (t_1/2α) = 0.05 h, t_1/2β = 0.3 h, maximum concentration (C_max)=148 ng/ml. It was demonstrated by NMR and mass spectrometry that cinobufagin was metabolized to desacetylcinobufagin and 3-epidesacetylcinobufagin in rats after intravenous injection. However, only 3-epidesacetylcinobufagin was detected in the serum of rats after oral administration of cinobufagin, indicating that cinobufagin is well absorbed in the gastrointestinal tract and prone to first-pass elimination. In addition, cinobufagin deacetylation was shown to occur in plasma and the liver.

With the advancement of modern science and technology, many new techniques have emerged, such as liquid chromatography-MS,^[99] UPLC-ESI-MS/MS,^[100] SPE-HPLC and^[101] high-speed counter-current chromatography.^[102] Thus, a more realistic and effective analysis of the pharmacokinetics of Chinese medicines can be performed. Liang et al. for the first time, established a sensitive, rapid and specific solid phase extraction high-performance liquid phase method (SPE-HPLC) to determine the pharmacokinetics of cinobufagin in rats after oral administration and obtained the following parameters: C_max = 0.77 ± 0.12 μg/ml, T_max = 20 ± 12 min, t_1/2 = 138 ± 30 min, apparent clearance (CL/F) = 0.093 ± 0.017 l/ (min kg), Vz/F = 18.045 ± 2.908 l/kg. More detailed data are presented in Table 2.

In 2010, Ning et al. used 2D-NMR as a basic method to isolate and identify nine metabolites of cinobufagin in rat bile and further demonstrated that hydroxylation is the main reaction involved in rat bile metabolism^.[104] After oral administration of cinobufagin to rats, 11 metabolites were obtained by liquid–liquid extraction, open column chromatography, medium pressure liquid chromatography and semi-preparative high-performance liquid chromatography. The main metabolic pathways of cinobufagin are hydroxylation, deacetylation and isomerization.^[105] Its metabolic pathways and metabolite structures are presented in Figure 4 and Table 3.

Biotransformation of natural products has great potential to generate new drugs, and the bioconversion of cinobufagin is possible using fungi of Mucor spinosus.^[106] Three biotransformation products were obtained and all showed cytotoxic activity in four tumour cell lines of Bel 7420, BGC 823, HeLa and HL 60.^[107] Besides, cunninghamella elegans^[108] and syncephalastrum racemosum^[109] have also been shown to be used for the biotransformation of cinobufagin, with the resulting transformation products possessing some biological activity and serving as a reference for the identification of cinobufagin metabolites in humans.

Conclusions and Perspectives

The prominent feature of cancer is its uncontrollable proliferation, which is a serious health hazard. Cinobufagin is derived from the dried secretions of the postauricular or cutaneous glands of Chinese toad or black orbital toad, which is one of the main active ingredients. A number of studies have demonstrated the efficacy of cinobufagin in the treatment of many cancers in vivo and in vitro, such as rectal cancer, colorectal cancer, oral squamous cell carcinoma, osteosarcoma, nasopharyngeal cancer, melanoma, cholangiocarcinoma, glioblastoma multiforme, hepatocellular carcinoma, gastric cancer, oesophageal squamous cell carcinoma, prostate cancer, breast cancer and cervical cancer. The mechanisms of action involved are also described in this article, cinobufagin can induce apoptosis through activation of the death receptor pathway and mitochondrial pathway, inhibit cancer cell proliferation, and block cell cycle in S-phase or G2/M-phase. In addition, it can also exert anticancer effects by inhibiting cancer angiogenesis, triggering DNA damage and relieving cancer pain. To further explore its anticancer mechanism, many scholars have investigated the metabolic process and anticancer effects of cinobufagin in vivo through pharmacokinetic analysis and the establishment of xenograft models. In recent years, good progress has been made regarding tumour immunotherapy. Cinobufagin upregulates the gene expression of antibacterial peptides (AMPs) hBD-2 and hBD-3 in dendritic cells, induces neutrophils to secrete HNP1-3 and hCAP-18/LL-37, and enhances the antibacterial activity of neutrophils, indicating that cinobufagin has a wide range of functions and further supports its anticancer potential.^[110]

The structure and hydrophobicity of cinobufagin and resibufogenin in crude extracts of toad venom are similar, so it is difficult to separate them. However, cyclodextrin as mobile phase additive could significantly improve the separation and purification of cinobufagin.^{[111, 112]} In addition, silica gel column chromatography with cyclohexane–acetone (5 : 1) as the mobile phase and high-speed counter-current chromatography (a two-phase solvent system consisting of n-hexane: chloroform: methanol: water (4 : 1 : 2.5 : 5 and 4 : 1 : 4 : 5, v/v)) can be used for the efficient purification of cinobufagin.^{[113, 114]} Given the limitations of natural resources, the artificial synthesis of cinobufagin needs to be considered, which is important for reducing the cost of new drug development and protecting natural resources. In addition, the target and anticancer mechanism of action for cinobufagin needs to be elucidated clearly. Although the toxic effects of cinobufagin in vivo are not obvious at pharmacological doses, the systematic safety evaluation is lacking such as acute toxicity, long-term toxicity and reproductive toxicity, and toxicokinetics should be further explored. Taking 120 patients with bone metastasis as the study object, the clinical efficacy of intravenous zoledronic acid combined with oral cinobufagin in the treatment of metastatic bone tumours was improved.^[115] Although experimental data for direct clinical use are relatively scarce, cinobufagin has the potential to be further developed as a new drug against cancer.

Author Contributions

HZ: conceptualization. C-LD and R-JZ: writing-original. C-LD, KR and HZ: writing-review and editing. HZ, PA and Y-QD: verification and recommendation. All authors contributed to the article and approved the submitted version.

Funding

This work was supported by funds from the National Natural Science Foundation of China (No. 82174023).

Ethical Statement

There are no ethical issues related to this work.

Conflict of Interest

The Authors declare that they have no conflict of interests to disclose.

Data Availability Statement

No new data were generated or analysed in support of this research.

References

Author notes