Search

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.

Current therapeutic strategies cannot eradicate hepatitis B virus covalently closed circular DNA (HBV cccDNA), which accounts for the persistence of HBV infection. Very recently, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been used as an efficient and powerful tool for viral genome editing. Given that the primary duck hepatocyte (PDH) infected with duck hepatitis B virus (DHBV) has been widely used to study human HBV infection in vitro, the present study aimed to demonstrate the targeted inhibition of DHBV DNA, especially cccDNA, by the CRISPR/Cas9 system using this model. We designed six single-guide RNAs (sgRNA1-6) targeting the DHBV genome. The sgRNA/Cas9 plasmid was transfected into DHBV-infected PDHs, and then DHBV total DNA (in culture medium and PDHs) and cccDNA were quantified by reverse transcription-quantitative polymerase chain reaction. The combined inhibition of CRISPR/Cas9 system and entecavir (ETV) was also assessed. Two sgRNAs, sgRNA4 and sgRNA6, exhibited efficient inhibition on DHBV total DNA (77.23 and 86.51%, respectively), cccDNA (75.67 and 85.34%, respectively) in PDHs, as well as DHBV total DNA in the culture medium (62.17 and 59.52%, respectively). The inhibition remained or enhanced from day 5 to day 9 following transfection. The combination of the CRISPR/Cas9 system and ETV further increased the inhibitory effect on DHBV total DNA in PDHs and culture medium, but not cccDNA. The CRISPR/Cas9 system has the potential to be a useful tool for the suppression of DHBV DNA.

Heme oxygenase‑1 (HO‑1), as an inducible and cytoprotective enzyme, has a protective effect against cellular oxidative stress. In the present study, cholesterol was used to induce lipid overload and increase reactive oxygen species (ROS), leading to oxidative stress in EA.hy926 cells. In the present study, western blotting and immunofluorescence analysis were used to detect the expression level of important molecules in the metabolism process of cholesterol. It was confirmed that cholesterol stimulation upregulated the expression of HO‑1 in a time‑dependent manner via the activation and translocation of nuclear factor erythroid 2‑related factor 2 (Nrf2), activation of the mitogen‑activated protein kinase (MAPK)/extracellular signal‑regulated kinase (ERK) signaling pathway and increasing intercellular Ca2+ ([Ca2+]i) concentration. The results showed that increasing the expression of HO‑1 decreased activation of the phosphoinositide 3‑kinase (PI3K)/AKT signaling pathway and inhibited the expression of c‑Myc. It was confirmed that cholesterol‑mediated oxidative damage in vascular endothelial cells induced an increase in the expression of HO‑1 via the activation of Nrf2 and the MAPK/ERK signaling pathway, and increasing the [Ca2+]i concentration. The overexpression of HO‑1 alleviated oxidative damage through inhibition of the PI3K/AKT signaling pathway and downregulation of the expression of c‑Myc.