Nutlin-3 reverses the epithelial-mesenchymal transition in gemcitabine-resistant hepatocellular carcinoma cells

Nutlin-3, a small molecule regulator of the tumor suppressor p53, targets the interaction between p53 and murine double minute 2 (MDM2) thereby promoting stabilization of p53 and subsequent p53-dependent induction of apoptosis and cell cycle arrest. Recent studies have demonstrated that Nutlin-3 plays a critical role in regulating tumor cell migration, invasion, metastasis, and drug resistance. Although these studies identified various biological functions of Nutlin-3, our understanding of the exact molecular mechanisms of Nutlin-3-mediated antitumor activity remains incomplete. In this study, we elucidated a role of Nutlin-3 in reversing the epithelial-mesenchymal transition (EMT) in gemcitabine-resistant (GR) hepatocellular carcinoma (HCC) cells. We assessed the effect of Nutlin-3 treatment on cell growth, migration, and invasion in both parental HCC cells and GR HCC cells. Moreover, we detected the expression of EMT markers in GR HCC cells treated with Nutlin-3 by real-time RT-PCR and western blot analysis, respectively. We found that Nutlin-3 inhibited cell migration and invasion in the GR HCC cells. Additionally, Nutlin-3 treatment increased E-cadherin protein levels, but decreased the protein levels of vimentin, Snail and Slug in the GR HCC cells. Furthermore, we found that Smad2 was highly expressed in the GR HCC cells compared with their parental HCC cells, and Nutlin-3 treatment downregulated Smad2 expression in the GR HCC cells. Depletion of Smad2 retarded cell migration and regulated the expression of EMT markers in GR HCC cells similarly to Nutlin-3 treatment. Our findings highlight an important role of Nutlin-3 in reversing EMT in GR cells through regulation of Smad2 expression, suggesting that Nutlin-3 could be a potential agent for the treatment of HCC patients with gemcitabine resistance.