Cinobufotalin inhibits the epithelial-mesenchymal transition of hepatocellular carcinoma cells through down-regulate β-catenin in vitro and in vivo

Hepatocellular carcinoma (HCC) is one of the malignant tumors with high incidence and mortality. The prognosis of HCC is poor due to the high postoperative recurrence rate and metastasis rate. Epithelial-mesenchymal transition (EMT) plays a key role in the metastasis of HCC, which is closely related to the invasion, intrahepatic metastasis and low survival rate. Here we demonstrated that cinobufotalin can upregulate epithelial markers (E-cadherin) and downregulate mesenchymal markers (N-cadherin, snail, slug and ZEB1) in HepG2, SMMC-7721 and SNU-368 cells. We further found that the mRNA and protein expression of β-catenin and its target genes (i.e. MMP7 and DKK1), which are related to tumor invasion and metastasis, were decreased after cinobufotalin treatment. Overexpression of β-catenin promoted EMT of HepG2 and SMMC-7721 cells, and cinobufotalin could antagonize this induction. While Knockdown of β-catenin could inhibit EMT and cinobufotalin enhanced this inhibition. In addition, cinobufotalin significantly suppressed the tumor EMT, as demonstrated by increased E-cadherin expression and decreased N-cadherin and vimentin expression, and inhibited formation and metastasis of lung metastases in vivo. In conclusion, our study has revealed a novel anticancer mechanism of cinobufotalin, which inhibits EMT progress by downregulating β-catenin, and then prevents the migration and invasion of HCC. These results provide convincing evidence for the development of cinobufotalin as a potential HCC metastasis inhibitor.