Re-expression of DIRAS3 and p53 induces apoptosis and impaired autophagy in head and neck squamous cell carcinoma.

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 re-expression 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 up-regulated 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.

In Vitro and Clinical Studies of Gene Therapy with Recombinant Human Adenovirus-p53 Injection for Malignant Melanoma.

Malignant melanoma is an aggressive tumor with high fatality rates and poor prognosis, mainly due to the lack of efficient treatment methods. The present study investigated the potential antitumor effects of recombinant adenovirus p53 (rAd-p53) on human malignant melanoma. The optimal viral titer on a human malignant melanoma (A-375) cell line was determined for the rAd-p53 treatment. The invasive abilities, apoptosis, variations in the cell cycle, and molecular expression levels of A-375 cells were detected after infection by rAd-p53. A tumor growth curve and hematoxylin and eosin staining were carried out for experiments in nude mice. Twenty-one patients with malignant melanoma were evaluated, including 12 cases without gene therapy and nine cases with rAd-p53 gene therapy. The overall survival rate and the median survival time were analyzed between the two groups of patients. When the multiplicity of infection was 100, the cells showed the best transfection efficiency. The invasive ability, apoptosis, cycle changes of the cells, and the expression of the p53, p21, and Bax genes and proteins were significantly changed in the experimental group. In nude mice, the tumor growth curve and the tumor size in the experimental group were significantly smaller than those of the control group. Hematoxylin and eosin staining revealed tumor metastasis in the blank group and the control group but not in the experimental group. Between the two groups of patients, the median survival of the gene therapy group (38 months) was greater than that of the group without gene therapy (27 months). In this study, high expression of the p53 gene could regulate the gene expression and reduce the invasive and metastatic abilities of the tumor cells. Furthermore, rAd-p53 effectively improved the survival of patients with malignant melanoma. Therefore, rAd-p53 may be a potential treatment method for human malignant melanoma.