Nutlin-3 Promotes TRAIL-Induced Liver Cancer Cells Apoptosis by Activating p53 to Inhibit bcl-2 and Surviving Expression.

ABSTRACT

OBJECTIVE: Tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL) is a potent anticancer agent, which could specifically target cancerous cells. Nutlin-3, a small-molecular inhibitor of murine double minute 2 (MDM2), shows oncogenic potential in a variety of human cancers. It has also been found to promote the TRAIL-induced apoptosis of cancer cells in esophageal squamous cancer, but its potential role and underlying mechanisms in the TRAIL-treated hepatocellular carcinoma (HCC) remains to be elucidated. METHODS: HSS cell line (Huh7) cells were used as an in vitro model of HCC. TRAIL (100 ng/ml) was used to induce cell apoptosis. Cell viability was measured by CCK-8 assay. Cell apoptosis was detected using TUNEL staining. Mitochondrial activity was evaluated by measuring caspase 3/7 and 9 levels. P53 expression at protein and mRNA level were measured by Western blotting and RT-qPCR, respectively. RESULTS: The combination of Nutlin-3 and TRAIL facilitated the apoptosis and increased the levels of mitochondrial cleaved-caspase3/7 and 9 in HCC cells compared with TRAIL treatment alone, both in a concentration-dependent way. Nutlin-3 also upregulated the expression of p53 and DR5, while knockdown of p53 significantly hindered the pro-apoptotic effect of Nutlin-3. Further studies revealed that Nutlin-3 downregulated the expression of survivin and bcl-2, both of which could be reversed by p53 knockdown. Moreover, survivin suppressant YM155 and bcl-2 inhibitor YM155 further enhanced the apoptosis of HCC cells in the presence of Nutlin-3 and TRAIL. CONCLUSION: These results suggested that Nutlin-3 facilitated TRAIL-induced apoptosis of HCC cells by activating the p53-survivin/bcl-2 pathway, which provided novel insights into the mechanism of Nutlin-3 and confirmed the potential of combination of Nutlin-3 and TRAIL as an adjuvant in HCC therapy.