COE inhibits vasculogenic mimicry in hepatocellular carcinoma via suppressing Notch1 signaling.ETHNOPHARMACOLOGICAL RELEVANCE
 Celastrus orbiculatus extract (COE), a mixture of 11 terpenoids isolated from the Chinese Herb Celastrus orbiculatus Vine, has been suggested to be effective in cancer treatment.


 In the current study, experiments were carried out to examine the effect of COE on VM formation and HCC tumor growth both in vitro and in vivo.


 CCK-8 assay and Nikon live-work station were used to observe the viability of malignant cells treated with COE.  Results showed that COE can inhibit HCC cells proliferation and invasion in a concentration-dependent manner.  VM formation induced by TGF-β1 was blocked by COE.  In mouse xenograft model, COE inhibited tumor growth and VM formation.  Both in vitro and in vivo studies showed that COE can downregulate expression of Notch1 and Hes1.


 The current results indicate that COE can inhibit VM formation and HCC tumor growth by downregulating Notch1 signaling.  This study demonstrates that COE is superior to other anti-angiogenesis agents and can be considered as a promising candidate in HCC treatment.
COE
CCK-8 assay and Nikon live-work station were used to observe the viability of malignant cells treated with COE. 
CCK-8 assay
CCK-8 assay and Nikon live-work station were used to observe the viability of malignant cells treated with COE. 
Nikon live-work station
Cell invasion was examined using Transwell. 
Transwell
Matrigel was used to establish a 3-D culture condition for VM formation. 
Matrigel
Changes of mRNA and protein expression were examined by RT-PCR and Western Blot respectively. 
RT-PCR
Changes of mRNA and protein expression were examined by RT-PCR and Western Blot respectively. 
Western Blot
Tumor growth in vivo was monitored using in vivo fluorescence imaging device. 
in vivo fluorescence imaging device
PAS-CD34 dual staining and electron microscopy were used to observe VM formation. 
PAS-CD34 dual staining
PAS-CD34 dual staining and electron microscopy were used to observe VM formation. 
electron microscopy
Immunohistochemical staining (IHC) was used to examine Notch1 and Hes1 expression in tumor tissues.



Immunohistochemical staining (IHC)