The effect of AkT-1 and JAk-1 silencing on the angiogenesis, proliferation and apoptosis in oral squamous cell carcinoma; in vitro and in vivo

Abstract

The aim of this study was to find a new therapeutic strategy targeting prominent molecules affecting HNSCC. Akt1 and Jak1 have key roles in apoptosis and proliferation of many cancers. In the present study, HNSCC cell line (HN5) and normal human cell line (HUVEC) were treated with Akt1 and Jak1 small interfering RNAs (siRNA). Consequently, the proliferation assessed by MTS and apoptosis by flow cytometry and DAPI tests. The genes expression and their relevant proteins were evaluated with ELISA and Real Time-PCR. The viability reduced to 64.57 % after treatment with Akt1 in HN5 cells. In HUVEC cell line, the most reduction of viability was seen in Akt1/Jak1/Cisplatin treated group which was 80.54%. Treatment of HN5 with Jak1/Cisplatin siRNA significantly increased the percentage of apoptotic cells (96.5%). In HUVEC cell line, Akt1/Jak1 siRNA treated group demonstrated the highest increased amount of apoptotic cells (5.31%). The Akt1 gene expression ratio in treated HN5 and HUVEC cells respectively decreased to 0.04, 0.03 (Jak1); 0.03, 0.02 (Jak1/cisplatin); 0.01, 0.04 (Akt1/Jak1); 0.08, 0.11 (Akt1/Jak1/cisplatin); 0.07, 0.02 (Akt1/cisplatin); and 0.01, 0.02 (Akt1) compared to untreated control group. The Jak1 gene expression ratio in treated cancerous HN5 and normal HUVEC cells respectively reduced to 0.2, 0.36 (Jak1); 0.029, 0.55 (Jak1/cisplatin); 0.28, 0.18 (Akt1/Jak1); 0.023, 0.70 (Akt1/Jak1/cisplatin); 0.01, 0.14 (Akt1/cisplatin); and 1.65, 0.24 (Akt1) in comparison with untreated control. In HUVEC cell line, the protein concentration of Akt1 in Akt1 and Cisplatin siRNA treated group was the least and there was no significant difference among other treated groups. In HN5 cells, the highest decrease was observed in the cells treated with Akt1 siRNA. In HUVEC cell line, Akt1 and Jak1 siRNA treated group most of all reduced Jak1 protein concentration. In HN5 cell line, Jak1 and cisplatin treated group induced the most reduction in Jak1 protein concentration while there was no significant difference among treated groups. In invivo assay, twenty male rats (six weeks old, n=5) were treated three times a week by dissolving 50 μL 4-NQO in distilled water. Intra-tumoral injection of siRNA-Hiperect was provided after HNSCC induction. The cumulative dose of injection was 2 nmol of siRNA and 360 µg of Hiperfect. Tumor size in all siRNA treated rats was reduced but the most reduction was seen in Akt1 treated rats (3 ± 0.3). Histopathological features of malignancy in the treatment of Akt1 siRNAs were least in H & E staining. In conclusion, knocking down Akt1 and Jak1 gene using siRNA significantly increases apoptosis and decreases proliferation in HNSCC cells regardless of the use of cisplatin.