| Kinase Assay: |
| 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. |
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| Cell Research: |
| 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. |
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Cell. 2018 Jan 11;172(1-2):249-261.e12. doi: 10.1016/j.cell.2017.12.019.IF=36.216(2019)
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