Effect of siRNA-mediated inhibition of nuclear factor-κB in combination with 5-FU on apoptosis of squamous cell carcinoma cell lines

Abstract

AIM: To evaluate the inhibitory effects of nuclear factor-kappa B p65(NF-κB p65)on expression of p65 and to determine its combinatory effects with 5-FU on proliferation and apoptosis in EC9706 and Eca109 esophageal squamous cell carcinoma (ESCC). METHODS: EC9706 and Eca109 cells were trans-fected with 50 nmol/L p65 siRNA. The expres-sion level of p65 mRNA was measured using PT-PCR at 0, 24, 48 and 72 h. P65 and Bcl-2 protein levels were determined using Western blotting. The cells were also stained with FITC-annexin V and PI, and cancer cell apoptosis was detected by flow cytometry using CellQuest acquisition and analysis programs. Morphological changes of ESCC cells were observed microscopically following p65 siRNA transfection with or without 5-FU. RESULTS: For EC9706 and Eca109 transfected with p65 siRNA, the expression level of the p65 mRNA in ESCC cells was down-regulated with time. Peak inhibitory effect occured at 72 h and a significant difference was detected, compared with 0 h (0.12±0.01 vs 0.28±0.05, 0.1±0.01 vs 0.38±0.04, both P＜0.05). The protein levels of p65 and Bcl-2 decreased after transfection with p65 siRNA at 72 h. There was a significant increase in apoptosis level at 72 h following p65 siRNA transfection (6.65%±0.27% vs 2.03%±0.08%, 8.03%±0.06% vs 2.66%±0.25%, both P＜0.05). The proliferation of EC9706 and Eca109 was slow after transfection with p65 siRNA at 72 h, while p65 siRNA in combination with 5-FU significantly inhibited cell proliferation. CONCLUSION: p65 siRNA can block NF-κB signaling pathway, down-regulates expression of Bcl-2, which makes activated NF-κB pathway a potential key target in gene therapy for ESCC.