Cinobufagin

Immunopharmacol Immunotoxicol. 2015 May 18:1-9.

Cinobufagin exerts anti-proliferative and pro-apoptotic effects through the modulation ROS-mediated MAPKs signaling pathway.[Pubmed: 25982794]

Cinobufagin (CBG) is a cardiotoxic bufanolide steroid secreted by the skin and parotid venom glands of the Asiatic toad Bufo bufo gargarizans (called Chan-Su). Although CBG is known to exhibit anti-cancer activities, very little is known about its potential mechanism(s) of action. In this study, we investigated whether CBG mediates its effect through the modulation of the mitogen-activated protein kinases (MAPKs) signaling pathway in human multiple myeloma (MM) U266 cells.
METHODS AND RESULTS:
We found that CBG caused the significant activation of ERK, JNK and p38 MAPK in U266 cells. CBG showed much higher cytotoxicity against U266 cells as compared to peripheral blood mononuclear cells (PBMC). Induction of CBG increased reactive oxygen species (ROS) generation from mitochondria, which is associated with the induction of apoptosis as characterized by increased sub-G1 DNA contents of cell cycle, positive Annexin V binding, activation of caspase-3 and cleavage of PARP. Inhibition of ROS generation by N-acetyl-l-cysteine (NAC) significantly prevented CBG-induced ERK, JNK and p38 MAPK activation and apoptosis. CBG also down-regulated the expression of various downstream gene products that mediate cell proliferation, survival, angiogenesis and metastasis. Interestingly, ERK, JNK and p38MAPK pharmacological inhibitors blocked CBG-induced MAPKs activation and ERK inhibitor (PD98059) also prevented the CBG-induced caspase-3 activation and PARP cleavage in U266 cells.
METHODS AND RESULTS:
Taken together, these findings suggest that CBG can act as a potent anticancer agent against MM and possibly exerts its effects through the ROS-mediated activation of ERK, JNK and p38 MAPK leading to the activation of caspase-3 in U266 cells.

Biol Pharm Bull. 2010;33(10):1728-32.

Anti-hepatitis B virus activities of cinobufacini and its active components bufalin and cinobufagin in HepG2.2.15 cells.[Pubmed: 20930383]

Cinobufacini (Huachansu) is a Chinese medicine prepared from the skin of Bufo bufo gargarizans Cantor (Bufonidae), which has long been used in traditional Chinese medicine (TCM). The aim of present study was to examine the anti-hepatitis B virus (HBV) activities of cinobufacini and its active components bufalin and Cinobufagin in the human HBV-transfected cell line HepG2.2.15.
METHODS AND RESULTS:
The hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and hepatitis B core-related antigen (HBcrAg) concentrations in cell culture medium were determined by chemiluminescent enzyme immunoassay after HepG2.2.15 cells were respectively treated with different concentrations of cinobufacini, bufalin, and Cinobufagin for 3 or 6 d. HBV DNA and mRNA were determined using transcription-mediated amplification and real-time polymerase chain reaction (PCR), respectively. On d 3, cinobufacini at a concentration of 1 μg/ml had no activity against HBV virological markers. However, on d 6, cinobufacini at 1 μg/ml effectively inhibited the secretion of HBsAg, HBeAg, and HBcrAg by 29.58, 32.87, and 42.52%. It was more potent than the positive control lamivudine (100 μg/ml). Bufalin and Cinobufagin slightly inhibited HBV antigen secretion. Treatment with cinobufacini, bufalin, or Cinobufagin had no anti-HBV effect on DNA in cell culture medium. Consistent with the HBV antigen reduction, HBV mRNA expression was markedly inhibited in comparison to the control when HepG2.2.15 cells were treated with cinobufacini, bufalin, or Cinobufagin.
CONCLUSIONS:
Results suggested that cinobufacini had more potent activity against HBV antigen secretion than its components bufalin and Cinobufagin and this inhibitory role was attributed to the specific inhibition of HBV mRNA expression.

Evid Based Complement Alternat Med. 2013;2013:851256.

A Study on the Mechanism of Cinobufagin in the Treatment of Paw Cancer Pain by Modulating Local β -Endorphin Expression In Vivo.[Pubmed: 24187573]

Cinobufagin has been widely used in the treatment of carcinoma and plays an important role in the relief of cancer pain. But the involved mechanism remains unknown. To investigate the changes in thermal and mechanical hyperalgesia in paw cancer pain in mice and the action mechanism of Cinobufagin using a paw cancer pain model.
METHODS AND RESULTS:
60 female mice were randomly divided into 5 groups: control group, model group, Cinobufagin group, Cinobufagin +NAL-M group, and morphine group; except ones in control group, mice were inoculated with H22 hepatoma cells in the right hind paw. From the 9th day after inoculation, mice were administrated drug once daily lasting for 8 days. The pain behavior was determined on the 2nd, 4th, 6th, and 8th days before and after administration. On the last day, they were sacrificed. The levels of β -END, CRF, and IL-1 β were analyzed by ELISA; immunohistochemistry was performed to detect the expressions of β -END, POMC, and μ -OR in the tumor and adjacent tissue. The thresholds of thermal pain and mechanical pain were significantly increased by Cinobufagin. Moreover, the expressions of β -END, CRF, POMC, and μ -OR were significantly upregulated by Cinobufagin. The analgesic effect of Cinobufagin was blocked by the peripheral opioid receptor antagonist NAL-M.
METHODS AND RESULTS:
Cinobufagin significantly relieved cancer pain in mice and raised their pain threshold, mainly upregulating the expression levels of β -END and μ -OR in the hind paw tumor and adjacent tissue.

PLoS One. 2014 Nov 24;9(11):e113272.

Resibufogenin and cinobufagin activate central neurons through an ouabain-like action.[Pubmed: 25420080]

Cinobufagin and resibufogenin are two major effective bufadienolides of Chan su (toad venom), which is a Chinese medicine obtained from the skin venom gland of toads and is used as a cardiotonic and central nervous system (CNS) respiratory agent, an analgesic and anesthetic, and as a remedy for ulcers. Many clinical cases showed that Chan su has severe side-effects on the CNS, causing shortness of breath, breathlessness, seizure, coma and cardiac arrhythmia.
METHODS AND RESULTS:
We used whole-cell recordings from brain slices to determine the effects of bufadienolides on excitability of a principal neuron in main olfactory bulb (MOB), mitral cells (MCs), and the cellular mechanism underlying the excitation. At higher concentrations, Cinobufagin and resibufogenin induced irreversible over-excitation of MCs indicating a toxic effect. At lower concentrations, they concentration-dependently increased spontaneous firing rate, depolarized the membrane potential of MCs, and elicited inward currents. The excitatory effects were due to a direct action on MCs rather than an indirect phasic action. Bufadienolides and ouabain had similar effects on firing of MCs which suggested that bufadienolides activated neuron through a ouabain-like effect, most likely by inhibiting Na+/K+-ATPase. The direct action of bufadienolide on brain Na+ channels was tested by recordings from stably Nav1.2-transfected cells. Bufadienolides failed to make significant changes of the main properties of Nav1.2 channels in current amplitude, current-voltage (I-V) relationships, activation and inactivation.
CONCLUSIONS:
Our results suggest that inhibition of Na+/K+-ATPase may be involved in both the pharmacological and toxic effects of bufadienolide-evoked CNS excitation.

Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2014 Mar;28(3):349-53.

Effects of cinobufagin on apoptosis in U-2OS osteosarcomas cells.[Pubmed: 24844018]

To investigate the effects of Cinobufagin on the apoptosis in U-2OS osteosarcomas cells (U-2OS cells) and explore its potential mechanism.
METHODS AND RESULTS:
The cytostatic effects of Cinobufagin (10, 20, 50, 100, 200, and 400 nmol/L) on U-2OS cells were evaluated by MTT assay at 24, 48, and 72 hours after culture; simple U-2OS cells served as control group. The impact of Cinobufagin (100 nmol/L) on the apoptosis in U-2OS cells was determined by flow cytometry at 48 hours after culture, which were treated with Cinobufagin (experimental group) or with Cinobufagin plus Z-VAD-FMK (control group), and simple U-2OS cells served as blank control group. The Caspase-3 activity was measured by Caspase-3 activity assay kit at 48 hours after culture, which were treated with Cinobufagin (20, 50, and 100 nmol/L), and simple U-2OS cells served as control group. The expression of apoptosis signal pathway related proteins in U-2OS cells treated with Cinobufagin were detected by Western blot at 48 hours after culture, which were treated with Cinobufagin (20, 50, and 100 nmol/L), and simple U-2OS cells served as control group. The results of MTT assay showed that Cinobufagin inhibited the proliferation of U-2OS cells in a dose- and time-dependent manners. At each time point, the growth rate of U-2OS cells was significantly reduced with the increasing Cinobufagin concentration, and as time prolonged, the growth rate of U-2OS cells behaved the same way in the same group. There were significant differences among different time points and groups (P < 0.05). The apoptotic rate of experimental group (46.87% +/- 11.23%) was significantly higher than that of the control group (2.34% +/- 0.98%) and blank control group (1.04% +/- 0.25%) (P < 0.05). The Caspase-3 activity in 20, 50, and 100 nmol/L groups were 1.14 +/- 0.32, 1.31 +/- 0.41, and 1.92 +/- 0.54, respectively, which were significantly higher than that in control group (P < 0.05). Compared with 20and 50 nmol/L groups, 100 nmol/L group significantly increased the Caspase-3 activity in U-2OS cells (P < 0.05). Compared with the control group, the expressions of cleaved Caspase-3, cleaved Caspase-9, and Bax were obviously up-regulated; the Bcl-2 expression was down-regulated; and the ratio of Bax/Bcl-2 was increased in different Cinobufagin-treated groups (P < 0.05). The same tendency was seen in different Cinobufagin-treated goups, showing significant differences among groups (P < 0.05).
CONCLUSIONS:
Cinobufagin can inhibite the proliferation of U-2OS cells, and induce cell apoptosis. The potential mechanism of Cinobufagin-induced apoptosis may be related to the mitochondria-mediated pathway.

Toxicol Lett. 2013 Apr 12;218(2):129-36.

The glycogen synthase kinase-3β/nuclear factor-kappa B pathway is involved in cinobufagin-induced apoptosis in cultured osteosarcoma cells.[Pubmed: 23164673]

Cinobufagin, a major component of cinobufacini (huachansu), is an important cardenolidal steroid. Several studies have suggested that Cinobufagin has potent anti-cancer effects. The present study examines the apoptosis-inducing activity and the underlying mechanism of action of Cinobufagin in osteosarcoma (OS) cells.
METHODS AND RESULTS:
Our results showed that Cinobufagin potently inhibited the proliferation of U2OS, MG63 and SaOS-2 cells. Significant increases in G2/M cell-cycle arrest and apoptosis in OS cells were also observed. The expression levels of several apoptotic proteins were assessed after Cinobufagin treatment in U2OS cells. Among them, xIAP, cIAP-1, survivin and Bcl-2 levels decreased remarkably, while the levels of Bax and cleaved-PARP increased. Furthermore, we validated the inhibition of GSK-3β/NF-κB signaling following Cinobufagin treatment. Western blots showed a decrease in nuclear p65 protein expression after exposure to different concentrations of Cinobufagin, while the phosphorylation of GSK-3β was simultaneously increased. Transduction with constitutively active forms of GSK-3β could protect against the downregulation of p65 and upregulation of cleaved-PARP that are induced by Cinobufagin treatment. However, combined treatment with Cinobufagin and SB216367 resulted in a significant reduction in p65 and an increase in cleaved-PARP in U2OS cells.
CONCLUSIONS:
Altogether, these results show that Cinobufagin is a promising agent for the treatment of OS. These studies are the first to reveal the involvement of the GSK-3β/NF-κB pathway in Cinobufagin-induced apoptosis.