Arenobufagin

Carcinogenesis. 2013 Jun;34(6):1331-42.

Arenobufagin, a natural bufadienolide from toad venom, induces apoptosis and autophagy in human hepatocellular carcinoma cells through inhibition of PI3K/Akt/mTOR pathway.[Pubmed: 23393227]

Hepatocellular carcinoma (HCC) is a deadly form of cancer without effective chemotherapy so far. Currently, only sorafenib, a multitargeted tyrosine kinase inhibitor, slightly improves survival in HCC patients.
METHODS AND RESULTS:
In searching for natural anti-HCC components from toad venom, which is frequently used in the treatment of liver cancer in traditional Chinese medicine, we discovered that Arenobufagin, a bufadienolide from toad venom, had potent antineoplastic activity against HCC HepG2 cells as well as corresponding multidrug-resistant HepG2/ADM cells. We found that Arenobufagin induced mitochondria-mediated apoptosis in HCC cells, with decreasing mitochondrial potential, as well as increasing Bax/Bcl-2 expression ratio, Bax translocation from cytosol to mitochondria. Arenobufagin also induced autophagy in HepG2/ADM cells. Autophagy-specific inhibitors (3-methyladenine, chloroquine and bafilomycin A1) or Beclin1 and Atg 5 small interfering RNAs (siRNAs) enhanced Arenobufagin-induced apoptosis, indicating that Arenobufagin-mediated autophagy may protect HepG2/ADM cells from undergoing apoptotic cell death. In addition, we observed the inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway by Arenobufagin. Interestingly, inhibition of mTOR by rapamycin or siRNA duplexes augmented Arenobufagin-induced apoptosis and autophagy. Finally, Arenobufagin inhibited the growth of HepG2/ADM xenograft tumors, which were associated with poly (ADP-ribose) polymerase cleavage, light chain 3-II activation and mTOR inhibition.
CONCLUSIONS:
In summary, we first demonstrated the antineoplastic effect of Arenobufagin on HCC cells both in vitro and in vivo. We elucidated the underlying antineoplastic mechanisms of Arenobufagin that involve cross talk between apoptosis and autophagy via inhibition of the PI3K/Akt/mTOR pathway. This study may provide a rationale for future clinical application using Arenobufagin as a chemotherapeutic agent for HCC.

J Chromatogr B Analyt Technol Biomed Life Sci. 2013 Nov 15;939:86-91.

Quantitative determination of arenobufagin in rat plasma by ultra fast liquid chromatography-tandem mass spectrometry and its application in a pharmacokinetic study.[Pubmed: 24113236]

A rapid, sensitive, and selective ultra fast liquid chromatography-tandem mass spectrometry method was developed for quantitative determination of Arenobufagin in rat plasma.
METHODS AND RESULTS:
Sample pretreatment involved a one-step protein precipitation with methanol using 0.1mL rat plasma. The separation was carried out on a Shim-pack XR-ODS II (75mm×2.0mm, i.d. 2.1μm) column with gradient elution at a flow rate of 0.30mLmin(-1). The mobile phase was acetonitrile and 0.1% formic acid in water. A post-column switching valve was applied to reduce the matrix effect. The detection was performed on a triple-quadruple tandem mass spectrometer in the multiple reaction monitoring mode after electrospray ionization. Linear calibration curves for Arenobufagin were obtained over the concentration range 1.056-1056ngmL(-1), with a lower limit of quantification of 1.056ngmL(-1). The intra-day and inter-day precision values were lower than 15% and the accuracy ranged from 5.4% to 9.8% at all quality control levels.
CONCLUSIONS:
The method was successfully applied to the determination and pharmacokinetic study of Arenobufagin in rat plasma following intraperitoneal administration.

Biochem Pharmacol. 2012 May 1;83(9):1251-60.

Arenobufagin, a bufadienolide compound from toad venom, inhibits VEGF-mediated angiogenesis through suppression of VEGFR-2 signaling pathway.[Pubmed: 22305746 ]

Angiogenesis is crucial for carcinogenesis and other angiogenic processes. Arenobufagin, one of the major components of toad venom, is a traditional Chinese medicine used for cancer therapy. It inhibits cell growth in several cancer cell lines. However, little is known about Arenobufagin's anti-angiogenic activity.
METHODS AND RESULTS:
In this study, we showed that Arenobufagin inhibited vascular endothelial growth factor (VEGF)-induced viability, migration, invasion and tube formation in human umbilical vein endothelial cells (HUVECs) in vitro. Arenobufagin also suppressed sprouting formation from VEGF-treated aortic rings in an ex vivo model. Furthermore, we found that Arenobufagin blocked angiogenesis in a matrigel plugs assay. Computer simulations suggested that Arenobufagin interacted with the ATP-binding sites of VEGFR-2 by docking. In addition, Arenobufagin inhibited VEGF-induced VEGFR-2 auto-phosphorylation and suppressed the activity of VEGFR-2-mediated signaling cascades.
CONCLUSIONS:
Taken together, our findings demonstrate that Arenobufagin is a specific inhibitor of VEGF-mediated angiogenesis.

Eur J Pharmacol. 1994 Feb 15;266(3):317-25.

Depressive effects of arenobufagin on the delayed rectifier K+ current of guinea-pig cardiac myocytes.[Pubmed: 8174614]

METHODS AND RESULTS:
The effects of a bufadienolide isolated from toad venom, Arenobufagin, a potent Na+/K+ pump inhibitor, were studied in single guinea-pig ventricular cells in the whole-cell patch-clamp configuration. Arenobufagin (50 microM) applied extracellularly decreased the amplitude of the delayed rectifier K+ current (IdK) by 30% without affecting the gating kinetics. The L-type Ca2+ current was also depressed, but to a lesser extent. The inward rectifier K+ current was hardly affected. Ouabain and the internal dialysis of cells with the solution containing 20 mM Na+ depressed IdK in a similar way as Arenobufagin. On the other hand, Arenobufagin also depressed IdK when the Na+/K+ pump was already inhibited in the K(+)-free Tyrode solution.
CONCLUSIONS:
Therefore, both a direct effect on the channel and an indirect effect through the inhibition of the Na+/K+ pump may be involved in the depression of IdK by Arenobufagin.

Yao Xue Xue Bao. 2011 May;46(5):527-33.

Anti-angiogenetic effect of arenobufagin in vitro and in vivo[Pubmed: 21800539 ]

This study is to investigate the anti-angiogenetic effect of Arenobufagin in vitro and in vivo.
METHODS AND RESULTS:
The anti-proliferation effect of Arenobufagin on CNE-2, Hep2, SH-SY5Y, LOVO, PC-3 and DU145 cells as well as human umbilical vein endothelial cells (HUVECs) was determined by MTT assay. Cell morphological changes of LOVO and HUVECs after Arenobufagin treatment were observed by microscopy. Arenobufagin inhibited the proliferation of CNE-2, Hep2, SH-SY5Y, LOVO, PC-3, DU145 and HUVECs in a dose-dependent manner. Furthermore, it was obviously observed that the subcytotoxic concentration of Arenobufagin in human carcinoma cells induced a marked decrease in the viability of HUVECs. Chick embryo chorioallantoic membrane (CAM) model was used to detect the anti-angiogenetic effect of Arenobufagin in vivo. Arenobufagin significantly suppressed the angiogenesis of CAM. Cell cycle analysis demonstrated that G2/M phase was arrested and the sub-G1 peak appeared with the increase of Arenobufagin concentration. PI/Annexin V double staining assay further demonstrated that Arenobufagin could induce apoptosis in a dose- and time-dependent manner. Mitochondrial potential collapse detected by flow cytometric analysis was increased after Arenobufagin treatment. It also observed that PARP was cleaved to p85 active form by Western blotting.
CONCLUSIONS:
Taken together, Arenobufagin has significant anti-angiogenetic effect in vitro and in vivo, and the action mechanisms behind its anti-angiogenesis may be associated with cell cycle arrest and apoptosis of vein endothelial cells.