Pharmacological Effects of Cinobufagin

Background

Toad venom (Bufonis Venenum, known as ‘Chansu’ in Chinese) has been used as a traditional Chinese medicine for more than a thousand years and is listed in the Chinese Pharmacopoeia (2020 edition) [1,2]. It is prepared from the dried white secretion of the auricular glands and the skin glands of Bufo gargarizans Cantor or Bufo melanostictus Schneider [3]. According to the theory of traditional Chinese medicine, it has efficacy in providing detoxification, pain relief, resuscitation, and refreshment [4,5]. The chemical constituents of toad venom mainly include bufadienolides, indole alkaloids, sterols, polysaccharides, amino acids, and organic acids, among which bufadienolides and indole alkaloids are considered to be the main active ingredients [6–8]. Modern pharmacological studies have demonstrated that toad venom possesses a multitude of pharmacological activities, including antitumor, cardiac, antibacterial, and analgesic activities effects [4,9,10].

Cinobufagin (CBF), a bufadienolide derived from toad venom, was first isolated by Jensen et al from Chansu in 1932 [11]. Multiple studies have focused on its pharmacological effects and molecular mechanisms. This suggests that CBF has broad prospects in therapeutic applications. However, at present there is no systematic review on CBF. This article reviews the pharmacological properties of CBF, including its antitumor, analgesic, cardioprotection, immunomodulatory, antifibrotic, antiviral, and antiprotozoal effects, and its physicochemical and pharmacokinetic properties. This article aims to review in vitro and preclinical studies on cinobufagin, a bufadienolide derived from toad venom.

Physicochemical Properties

As shown in Figure 1, CBF is a bufadienolide with properties such as molecular structure[(1R,2S,4R,5R,6R,7R,10S,11S,14S,16R)-14-hydroxy-7,11-dimethyl-6-(6-oxopyran-3-yl)-3-oxapentacyclo [8.8.0.02,4.02,7.011,16] octadecan-5-yl] acetate, molecular formula: C26H34O6, molecular weight of 442.54 g/mol, and melting point of 222–223°C [12–14].

Pharmacological Activities of CBF

ANTITUMOR EFFECTS OF CBF:

Cancer is a common disease globally that seriously affects human health. Recent studies have illuminated that CBF suppressed the growth of a wide variety of tumor cells, including liver cancer, osteosarcoma, melanoma, colorectal cancer, acute promyelocytic leukemia, nasopharyngeal carcinoma, multiple myeloma, breast cancer, and gastric carcinoma [15]. The anticancer activities and related mechanisms of CBF are listed in Table 1.

LIVER CANCER:

Liver cancer is a common digestive malignant cancer worldwide with a high mortality rate. Several studies have shown that CBF can inhibit proliferation of hepatocellular carcinoma cells. Wendong Feng et al used CCK-8 assay to examine the growth of cells treated with CBF [16]. The results showed that when CBF concentration increased from 0 to 320 ng/l, the inhibition of HepG2 cells increased from 0% to 65% at 12 h, to 87% at 24 h, and to 99% at 48 h, and the LD50 of CBF was estimated to be 170, 78, and 40 ng/L, respectively. These data indicated that CBF inhibits the proliferation of HepG2 cells in a concentration- and time-dependent manner and has cytotoxicity to HepG2 cells even at very low concentrations. CBF has been found to induce HepG2 cells apoptosis. The apoptotic rate of HepG2 cells treated with CBF (100 ng/L) for 12 h, and 24 h were 13.6% and 25.5%, respectively, whereas the apoptotic rate of HepG2 cells in the control group was 1.6% and 3.2%, respectively. Moreover, CBF reduced the migration ability of HepG2 cells. Liang et al reported that CBF could inhibit HepG2 cell proliferation in vitro (IC50 values were 86.025 μM) and tumor growth in vivo [11]. Xiaohan Jin et al demonstrated that viability rates of Huh-7 cells treated with 1 μmol/L CBF for 12, 24, and 36 h were 74.5±4.5%, 53.7±3.8%, and 27.9±2.3%, respectively, in vitro and CBF induced cell cycle G2/M phase arrest in Huh-7 cells. CBF also suppressed the growth of Huh-7 tumors in vivo when CBF was intratumorally injected at a 10 mg/kg dose for 21 days [17].

Mechanistic studies indicated that CBF could inhibit proliferation and induce apoptosis of HepG2 cells by upregulating the p53 pathway and downregulating the Akt and ERK pathways [16]. In addition, CBF triggers defects in spindle formation and cap-dependent translation in liver cancer cells by inhibiting the AURKA-mTOR-eIF4E axis [17].

OSTEOSARCOMA:

Osteosarcoma is the most common primary malignant bone tumor. CBF has been reported to inhibit cell proliferation in osteosarcoma cells, including U2OS, MG63, and SaOS-2, in a time- and dose-dependent manner [18]. Researchers found that CBF induced cell cycle arrest at G2/M phases and apoptosis in U2OS, MG63, and SaOS-2 cells. Interestingly, toxicity studies have suggested that CBF has less toxicity or no toxicity to the human osteoblast cell line hFOB 1.19.

Researchers have conducted several studies on the potential mechanism of CBF for osteosarcoma therapy. Junqiang Yin et al found CBF-induced apoptosis via inhibition of the GSK-3β/NF-κB pathway [18]. Chuan Zhang et al demonstrated that CBF suppresses the characteristics of osteosarcoma cancer cells by inhibiting the IL-6-OPN-STAT3 pathway [19]. Kun Ma et al proved that CBF triggered apoptosis and autophagic cell death via activation of the ROS/JNK/p-38 axis [20].

MELANOMA:

Malignant melanoma is the deadliest type of skin cancer. CBF inhibited human malignant melanoma A375 cells proliferation (IC50=0.2 μg/mL, 24h) and cell colony formation. Additional studies demonstrated that CBF markedly increased the levels of ATM serine/threonine kinase (ATM) and checkpoint kinase 2 (Chk2) and decreased the levels of cell division cycle 25C (CDC25C), cyclin-dependent kinase 1 (CDK1), and cyclin B, subsequently inducing G2/M cell cycle arrest in A375 cells [21]. Moreover, CBF induced A375 cells apoptosis by inhibiting the PI3K/Akt pathway [21]. CBF also induced cell cycle arrest at the G2/M phase and cell apoptosis in B16 cells [21]. In addition, CBF exerted cytotoxicity against human uveal melanoma OCM1 cells (IC50= 0.8023 μM, 48 h) and inhibited the growth of OCM1 xenograft tumors in nude mice [22]. Researcher found that CBF arrested the cell cycle in the G1 phase in a concentration-dependent manner in OCM1 cells and induced cell apoptosis of OCM1 by activating the mitochondrial apoptotic signal pathway [22].

COLORECTAL CANCER:

Colorectal cancer is a common malignant tumor of the digestive system. CBF has been reported to inhibit the proliferation, migration, and invasion and also promote apoptosis of colorectal cancer cell lines (HCT116, RKO, and SW480 cells) [23]. In vivo, assays confirmed CBF inhibited the growth of HCT116 xenograft tumors in nude mice [23]. CBF also promoted apoptosis and inhibited the invasion, metastases, and angiogenesis of colorectal cancer cells [23, 24]. In addition, CBF demonstrated a strong reversal effect of multidrug resistance in P-gp-overexpressing LoVo/ADR, HCT116/L, and Caco-2/ADR cells, but did not affect their parental cells [25]. It also enhanced the effect of DOX against P-gp-overexpressing LoVo/ADR cell xenografts in nude mice [25].

Molecular mechanistic studies have illustrated CBF suppressed colorectal cancer growth via STAT3 pathway inhibition [23]. CBF also suppressed tumor neovascularization by disrupting the endothelial mTOR/HIF-1α pathway to trigger ROS- mediated vascular endothelial cell apoptosis [24]. The specific mechanism behind the reversal of drug resistance by CBF was also investigated. The results showed that CBF inhibited the activity of the P-gp ATPase but without altering the expression of P-gp [25].

ACUTE PROMYELOCYTIC LEUKEMIA:

Acute promyelocytic leukemia is a hematopoietic malignancy characterized by the accumulation of large amounts of immature blood cells in hematopoietic tissues. Studies have revealed that CBG inhibited the viability of NB4 and NB4-R1 cells in a time-and dose-dependent manner [26], and it induced NB4 and NB4-R1 cell apoptosis and PML-RARA degradation in a caspase-dependent manner by inhibiting the β-catenin signaling pathway.

NASOPHARYNGEAL CARCINOMA:

Nasopharyngeal carcinoma is an epithelial carcinoma arising from the nasopharyngeal mucosal lining. CBF significantly inhibited the proliferation of HK-1 cells [27]. Further analyses demonstrated that CBF induces cell cycle arrest at the S phase in HK-1 cells by downregulating the levels of CDK2 and cyclin E. Moreover, CBF downregulated the protein level of Bcl-2 and upregulated the levels of Bax, subsequently increasing the levels of cytoplasmic cytochrome c, Apaf-1, cleaved PARP1, cleaved caspase-3, and cleaved caspase-9, leading to HK-1 apoptosis. Furthermore, CBF increased ROS levels and decreased the mitochondrial membrane potential in HK-1 cells.

In addition, CBF inhibited 5–8F and HONE-1 cells proliferation and decreased the metastasis of 5–8F cells in vivo [28]. Molecular analysis revealed that CBF induced ENKUR to repress the β-catenin/c-Jun/MYH9 signal and thus decreased UBE3A-mediated p53 ubiquitination degradation. As a result, the epithelial-mesenchymal transition signal was inactivated to suppress nasopharyngeal carcinoma metastasis [28].

MULTIPLE MYELOMA:

Multiple myeloma is a clonal B-cell neoplasia. Compared to peripheral blood mononuclear cells, CBF showed much higher cytotoxicity against multiple myeloma U266 cells [29]. Mechanistic studies have demonstrated that CBF suppressed multiple myeloma growth through modulation of the ROS-mediated MAPKs signaling pathway.

OTHER CANCERS:

Some scholars have studied other antitumor activities of CBF. Xuanxuan Xiong et al found that CBF inhibited gastric cancer SGC-7901 cell proliferation and induced caspase-mediated apoptosis [30]. The IC50 value of CBF treatment at 24 h was 0.24 mM. Ling Zhu et al determined the inhibitory effect of CBF on MCF-7 cells [31], showing that CBF inhibited the growth of breast cancer cell lines MCF-7 in a time- and dose-dependent manner, and the IC50 values at 24, 48, and 72 h after treatment were 0.94±0.08, 0.44±0.12, and 0.22±0.03 μM, respectively.

Analgesic Effects of CBF

CBF had been widely used in the treatment of carcinoma and played an important role in the relief of cancer pain. It has been reported that CBF was better than morphine in the treatment of cancer pain. Although morphine works immediately, CBF has an effect on peripheral opioid receptors, has no adverse effects such as addiction like morphine and other opioids, and can be used as a substitute for morphine in the treatment of patients with cancer pain. The underlying mechanism responsible for the analgesic action of CBF was associated with an improved level of peripheral β-END and the role of peripheral opioid receptors [32]. Tao Chen et al found that CBF relieved cancer pain in mice and the CBF-induced local analgesic effect might be associated with increased activity of the POMC/β-END/μ-OR pathway released from invaded CD3/4/8 lymphocytes in cancer tissues [33]. Longsheng Xu demonstrated that CBG exerted significant antinociceptive effects in thermal and chemical pain models, possibly via activating 7nAChR, thereby triggering inhibition of the NF-κB signaling pathway [34].

Cardiotonic Effects of CBF

The effect of CBF on experimentally induced heart failure caused by acute local ischemia was examined through ligation of the left anterior descending coronary artery in a perfused guinea pig heart. The results demonstrated that CBF (3×10−7 M) restored coronary flow in the perfused guinea-pig heart to 90% of the pre-ligation level. Cardiac output and left ventricular pressure in the perfused heart increased to levels prior to the occurrence of acute local ischemia, and left ventricular work was enhanced by CBF (3×10−7 M) to 108% of the pre-ligation level. These findings suggest that CBF exhibits potent cardiotonic action in experimentally induced heart failure resulting from acute local ischemia [35]. Furthermore, researchers have discovered that CBF can inhibit Na+-K+-ATPase activity, shorten the action potential duration, and increase cardiac contractility in guinea pig hearts [36].

Immunomodulatory Effects of CBF

Scholars have found that CBF had potential immune system regulatory effects. They found that CBF stimulated cell proliferation of splenocytes and peritoneal macrophages and markedly enhanced the phagocytic activation of macrophages. CBF also significantly increased CD4+CD8+ double-positive T-cell populations and the percentage of S-phase cells of splenic lymphocytes and increased the ratio of Th1/Th2 [37,38]. Another study showed that CBF enhanced the protective efficacy of formalin-inactivated Salmonella typhimurium vaccine through Th1 immune response [39].

Antifibrotic Effects of CBF

CBF was reported to suppress fibroblast activation and differentiation, epithelial-mesenchymal transition, and, eventually, the extracellular matrix deposition by inhibiting the TGF-β1/Smad3 signaling. In vivo, experiments also proved that CBF attenuated bleomycin-induced pulmonary fibrosis in mice [40].

Antiviral Effects of CBF

The emergence of severe acute respiratory infectious diseases is often attributed to zoonotic beta coronaviruses such as SARS-CoV, MERS-CoV, and SARS-CoV-2 [41]. As reported by Jin et al, CBF showed high anti-MERS-CoV activities (IC50, 0.017 μM) and the selectivity indices (SI, CC50/IC50) were >564.7 for CBF [42]. CBF could be a promising bufadienolide for therapeutic use against emerging coronavirus infections such as COVID-19.

CBF was found to inhibit enterovirus 71(EV71) infection in vitro in cell viability and plaque reduction assays. The IC50 of CBF was (10.94±2.36) nmol/L, the 50% cytotoxic concentration (CC50) of CBF was (1277±223) nmol/L, and the anti-EV71 selectivity index (SI50) of CBF was 116.7, suggesting promise for drug development. Researchers found that CBF disrupted the synthesis of EV71 protein, but did not affect EV71 RNA replication. Hence, CBF may be a promising candidate for the treatment of EV71-caused disease [43].

Antiprotozoal Effects of CBF

Bio-guided studies have revealed the notable antimicrobial and antiparasitic properties of bufadienolides [44]. The antitrypanosomal activity of CBF was tested in vitro against T. cruzi. The results showed that CBF displayed low mammalian cytotoxicity (IC50= 29.0±2.57) [45].

The Pharmacokinetics of CBF

Pharmacokinetic studies are an important aspect of drug evaluation. Currently, there are limited pharmacokinetic data available on CBF. Wenlong Wei et al reported the pharmacokinetics of CBF in rats [46]. A method was established for rapidly and accurately determining the concentration of CBF in plasma after intragastric administration in a dose of 20 mg/kg in rats, and its specificity, accuracy, precision, recovery, matrix effect, and stability were studied. The Cmax, the time to Cmax (Tmax), and the t1/2 of CBF were 45.83±4.56 ng/mL, 0.083±0 h, and 2.79±0.93 h, respectively. This result indicated that CBF was absorbed and eliminated quickly. The main metabolite was desacetylcinobufagin. The Cmax and area under the curve (AUC0-t) of desacetylcinobufagin were much higher than CBF in plasma samples. However, there are few studies about the pharmacological actions of desacetylcinobufagin.

Future Directions

In this review, we provide a summary of the physicochemical, pharmacodynamic, and pharmacokinetic characteristics of CBF to establish a basis for clinical research. According to reports, CBF has been found to possess multiple pharmacological activities, including antitumor, analgesic, cardiotonic, immunomodulatory, antifibrotic, antiviral, and antiprotozoal effects. While there have been numerous studies on its anticancer activity, there is a relative scarcity of research regarding its other pharmacological effects. CBF has demonstrated therapeutic effects on various types of cancer, such as liver cancer, osteosarcoma, melanoma, colorectal cancer, acute promyelocytic leukemia, nasopharyngeal carcinoma, multiple myeloma, gastric cancer, and breast cancer. However, most of the research focusing on its antitumor activity has been limited to the cellular level, necessitating the need for more in vivo and clinical trial investigations. Additionally, the direct targets and mechanism of action of CBF have yet to be fully elucidated. A comprehensive understanding of the targets will aid in guiding clinical treatment strategies involving CBF. Combining CBF with traditional chemotherapy drugs is considered an optimal option for reducing drug resistance and improving cancer treatment outcomes. Nevertheless, there are few reports on the combined treatment of cancers with CBF and traditional chemotherapy drugs. The toxicity of CBF has raised significant concerns regarding its safety as a drug candidate. However, there is a dearth of studies investigating the toxicological and pharmacokinetic aspects of CBF.

Despite possessing numerous pharmacological activities, CBF exhibits poor aqueous solubility, leading to low bioavailability, thus limiting its clinical application [7]. Structural modification is a potential approach to improve CBF’s solubility. However, such modifications may also impact its bioactivity. Developing new pharmaceutical formulations is another way to enhance the bioavailability and solubility of CBF. Various techniques, including solid dispersion, microemulsion, submicroemulsion, cyclodextrin inclusion, and nanodrug delivery systems, have been employed to improve solubility [47,48]. Nevertheless, these methods still face limitations, such as the unresolved instability issues of liposomes, such as aggregation, fusion, and drug leakage during storage. Moreover, the safety concerns associated with these materials hinder their progress in clinical application.

Conclusions

CBF holds great potential as a versatile drug with various pharmacological activities. Further research encompassing in-depth analytical studies, identification of direct binding targets, pharmacokinetic and toxicological investigations, and development of new strategies to overcome its poor solubility are necessary to advance the development and clinical application of CBF.