RNA Interference Of NF-κB P65、Signal Transducers And Activators Of Transcription-3 (STAT-3) Enhances The Antitumor Effects Of Nimotuzumab MAb In Hypopharyngeal Carcinoma Fadu Cells

BackgroundThe development of head and neck squamous cell carcinoma (HNSCC) involves multiple genetic alterations in tumor-suppressor proteins, together with the persistent activation of growth-promoting signaling pathways. The epidermal growth factor receptor (EGFR) pathway is a most frequently studied in HNSCC. Additionally, overexpression of EGFR correlates with short survival in patients with HNSCC. Agents for EGFR targeting therapy or test are:anti-EGFR monoclonal antibodies and small molecule tyrosine kinase inhibitors (TKIs). Despite the overall survival improvement of EGFR targeting therapy, treatment outcomes of advanced and recurrent disease remain poor. Although mechanisms of treatment resistance are still unclear, over-activivation of downstream pathways is one of the mechanisms. In this study we examined the combined inhibition of downstream NF-κB and STAT-3 pathway to enhance anti-EGFR monoclonal antibody nimotuzumab induced hypopharynx fadu cell apoptosis and proliferation arrest.ObjectivesOur study was to investigate comination inhibition of NF-kB p65 and STAT-3 to enhance nimotuzumab in the proliferation and apoptosis of the hypopharyngeal carcinoma Fadu cells.Methods1 The EGFR, NF-κB p65 and STAT-3 in Fadu cell line were detected by western blot analysis after 72 hours incubation with indicated concentration of (Oug/mL,50 ug/mL, ug/mL,100 ug/mL,200 ug/mL) of nimotuzumab.Four interference target sites of NF-κB p65 and STAT-3 are selected to construct four groups of shRNA recombinant eukaryotic expression vector. A negative control vector is also constructed. ShRNA are transfected to 293T cell lines by liposomes. ShRNA that has significant interference is selected.2 After transfection, cells were divided into five groups:① lipopolysaccharide (LPS) (1 ug/mL)+shRNA p65; ② LPS (1 ug/mL)+shRNA STAT3;③LPS (1 ug/mL)+shRNAp65、STAT3; ④LPS (1 ug/mL)+shRNA negative control (NC); ⑤shRNA p65、STAT-3+nimotuzumab (100 ug/mL). The protein levels of EGFR, p65, STAT3 in the cells were detected by Western blotting at 48 hours after LPS stimulation. Cell proliferation, apoptosis and cell cycles were evaluated by flow cytometry at 48,72,96 hours.Results1 Nimotuzumab down-regulated level of EGFR in hypopharyngeal carcinoma fadu cells and up-regulated expressions of NF-κB p65 and STAT-3 in a dose-dependent manner. The result of DNA sequencing confirmed that recombinant plasmid of pGPU6/GFP/Hygro-p65, pGPU6/GFP/Neo-STAT-3 and pGPU6/GFP/NC were suceefully construced. Compare with mRNA of NC group, gene silencing of NF-κB p65 and STAT-3 was decreased to 53.2% and 47.4%, respectively.2 NF-κB p65, STAT-3 shRNA effectively down-regulated the protein levels of p65, STAT-3, respectively (p<0.05). Combination transfection was more effective to inhibit the proliferation and promote apoptosis of FaDu cells (p<0.01). Combination gene inhibition and nimotuzumab increased apoptosis and induced cell cycle arrest with the comparasion with combined gene siliencing group (p <0.01).ConclusionsCombination interfering expressions of NF-κp65 and STAT3 could significantly inhibit proliferation of FaDu cells, indicating that NF-κB p65, STAT3 may be used for further investigation of gene therapy of hypopharyngeal carcinoma and could enhance the antitumor effects of nimotuzumab.