摘要Background:p53 and DIRAS3 are tumor suppressors that are frequently silenced in tumors.In this study,we sought to determine whether the concurrent re-expression of p53 and DIRAS3 could effectively induce head and neck squamous cell carcinoma (HNSCC) cell death.Methods:CAL-27 and SCC-25 cells were treated with Ad-DIRAS3 and rAd-p53 to induce re-expression of DIRAS3 and p53 respectively.The effects of DIRAS3 and p53 re-expression on the growth and apoptosis of HNSCC cells were examined by TUNEL assay,flow cytometric analysis and MTT.The effects of DIRAS3 and p53 re-expression on Akt phosphorylation,oncogene expression,and the interaction of 4E-BP1 with eIF4E were determined by real-time PCR,Western blotting and immunoprecipitation analysis.The ability of DIRAS3 and p53 re-expression to induce autophagy was evaluated by transmission electron microscopy,LC3 fluorescence microscopy and Western blotting.The effects of DIRAS3 and p53 re-expression on HNSCC growth were evaluated by using an orthotopic xenograft mouse model.Results:TUNEL assay and flow cytometric analysis showed that the concurrent re-expression of DIRAS3 and p53 significantly induced apoptosis (P＜0.001).MTT and flow cytometric analysis revealed that DIRAS3 and p53 reexpression significantly inhibited proliferation and induced cell cycle arrest (P＜0.001).Mechanistically,the concurrent re-expression of DIRAS3 and p53 down-regulated signal transducer and activation of transcription 3 (STAT3)and upregulated p21WAF1/CIP1 and Bax (P＜0.001).DIRAS3 and p53 re-expression also inhibited Akt phosphorylation,increased the interaction of eIF4E with 4E-BP1,and reduced the expression of c-Myc,cyclin D1,vascular endothelial growth factor (VEGF),fibroblast growth factor (FGF),epidermal growth factor receptor (EGFR) and Bcl-2 (P＜0.001).Moreover,the concurrent re-expression of DIRAS3 and p53 increased the percentage of cells with GFP-LC3 puncta compared with that in cells treated with control adenovirus (50.00％±4.55％ vs.4.67％±1.25％,P＜0.001).LC3 fluorescence microscopy and Western blotting further showed that DIRAS3 and p53 re-expression significantly promoted autophagic activity but also inhibited autophagic flux,resulting in overall impaired autophagy.Finally,the concurrent re-expression of DIRAS3 and p53 significantly decreased the tumor volume compared with the control group in a HNSCC xenograft mouse model[(3.12±0.75)mm3 vs.(189.02±17.54)mm3,P＜0.001].Conclusions:The concurrent re-expression of DIRAS3 and p53 is a more effective approach to HNSCC treatment than current treatment strategies.