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The Na+/K+-ATPase inhibitor cinobufagin exhibits numerous anticancer effects on hepatocellular carcinoma (HCC) cells expressing wild-type p53 via inhibition of aurora kinase A (AURKA) and activation of p53 signaling. However, the effects of cinobufagin on HCC cells expressing mutant p53 remain unclear. In the present study, the anticancer effects of cinobufagin were investigated on HCC Huh-7 cells with mutant p53, and the effects of AURKA overexpression or inhibition on the anticancer effects of cinobufagin were analyzed. Viability, cell cycle progression and apoptosis of cells were determined using an MTT assay, flow cytometry and Hoechst 33342 staining, respectively. The expression levels of p53 and p73 signaling-associated proteins were investigated via western blot analysis. The results demonstrated that the expression levels of AURKA, B-cell lymphoma 2 (Bcl-2), cyclin-dependent kinase 1, cyclin B1, proliferating cell nuclear antigen and heterogeneous nuclear ribonucleoprotein K, as well as the phosphorylation of p53 and mouse double minute 2 homolog, were significantly decreased in Huh-7 cells treated with 5 µmol/l cinobufagin for 24 h. Conversely, the expression levels of Bcl-2-associated X protein, p21, p53 upregulated modulator of apoptosis and phorbol-12-myristate-13-acetate-induced protein 1, were significantly increased by cinobufagin treatment. Overexpression or inhibition of AURKA suppressed or promoted the anticancer effects of cinobufagin on Huh-7 cells, respectively. These results indicated that cinobufagin may induce anticancer effects on Huh-7 cells via the inhibition of AURKA and p53 signaling, and via the activation of p73 signaling, in an AURKA-dependent manner.

Cinobufagin is a Na+/K+-ATPase (NKA) inhibitor with excellent anticancer effects to prolong the survival of patients. The purpose of the present study was to clarify the underlying mechanism of the anticancer effects of cinobufagin using overexpression or inhibition of aurora kinase A (AURKA) signaling. First, high expression of Na+/K+-ATPase alpha 1 subunit (ATP1A1) and AURAK resulted in increased malignant transformation in hepatocellular carcinoma (HCC) patients using the cancer genome atlas (TCGA) data and tissue samples. After treatment with cinobufagin, we successfully screened 202, 249, and 335 changing expression proteins in Huh-7 cells under normal, overexpression, and inhibition of AURKA using tandem mass tags (TMT)-labeled quantitative proteomics coupled to 2D liquid chromatography-tandem mass spectrometry (LC-MS/MS). Bioinformatics analysis revealed that these molecules were closely associated with chromosome segregation, DNA damage, and regulation of translation processes. We further confirmed that cinobufagin induced DNA damage and chromosome segregation disorders and suppresses translational processing in oncogenes by decreasing the expression of AURKA, mechanistic target of rapamycin kinase (mTOR), p-mTOR, p-extracellular regulated protein kinases (ERK), eukaryotic translation initiation factor 4E (eIF4E), and p-eIF4E, while increasing the expression of p-eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) (S65, T37, T46, T45) and increasing the interaction between eIF4 and 4E-BP1. Our results suggested that cinobufagin performed an antitumor effects in liver cancer cells by inhibiting the AURKA-mTOR-eIF4E axis.