The small-molecule inhibitors of p53-murine double minute 2 interaction, such as Nutlin-3, are effective against cancers bearing wild-type p53. However, murine double minute 2 inhibitors often are unable to completely eliminate solid tumor cells. To address this issue, we investigated the anticancer effects of Nutlin-3 in combination with Oridonin in osteosarcoma cells. We found that Oridonin at sub-toxic concentrations synergistically enhanced Nutlin-3-mediated cell viability inhibition in wild-type p53 U2OS and SJSA-1, but not in p53-mutant MNNG/HOS and in null-p53 Saos-2 osteosarcoma cell lines. Importantly, in the presence of Oridonin, Nutlin-3 could completely abolish cell viability in the wild-type p53 osteosarcoma cell lines. Western blotting analysis showed that Oridonin treatment rapidly and distinctly increased the levels of all three forms of Bim and also markedly reduced the levels of Bcl-2 and Bcl-xl in osteosarcoma cells. Western blotting analysis further showed that Oridonin considerably enhanced Nutlin-3-triggered activation of caspases-9 and -3 and poly(ADP-ribose) polymerase cleavage. Flow cytometry assay showed that Oridonin significantly enhanced Nutlin-3-mediated apoptosis in wild-type p53 osteosarcoma cells. Overall, our results suggest that the combined treatment of Nutlin-3 plus Oridonin may offer a novel therapeutic strategy for osteosarcoma.

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Abstract. The present study aimed to identify the genes directly or indirectly correlated with the amplification of MYCN in neuroblastoma (NB). Microarray data (GSE53371) were downloaded from Gene Expression Omnibus, and included 10 NB cell lines with MYCN amplification and 10 NB cell lines with normal MYCN copy numbers. Differentially expressed genes (DEGs) were identified using the Linear Models for Microarray Data package, and a false discovery rate of <0.05 and |log 2 FC (fold change)|>1 were selected as cut-off criteria. Hierarchical clustering analysis and Gene Ontology analysis were respectively performed for the DEGs using the Pheatmap package in R language and The Database for Annotation, Visualization and Integrated Discovery. A protein-protein interaction network (PPI) was constructed for the DEGs using the Search Tool for the Retrieval of Interacting Genes database. Pathway analysis was performed for the DEGs in the PPI network using the WEB-based GEne SeT AnaLysis Toolkit. The correlation between MYCN and the key gene associated with MYCN was determined using Pearson's correlation coefficient. In total, 137 downregulated and 35 upregulated DEGs were identified. Functional enrichment analysis indicated that KCNMB4 was involved in the regulation of action potential in neuron term, and the FOS, GLI3 and GLI1 genes were involved in the extracellular matrix-receptor interaction pathway. The PPI network and correlation analysis revealed that the expression of SOX2 was directly correlated with the expression of MYCN, and the correlation coefficient of SOX2 and MYCN was -0.83. Therefore, SOX2, KCNMB4, FOS, GLI3 and GLI1 may be involved in the pathogenesis of NB, with the expression of SOX2 downregulating the expression of MYCN.