Nuclear Factor-Kappa B Inhibition Can Enhance Apoptosis of Differentiated Thyroid Cancer Cells Induced by 131I

Abstract

To evaluate changes of nuclear factor-kappa B (NF-κB) during radioiodine 131 (¹³¹I) therapy and whether NF-κB inhibition could enhance ¹³¹I-induced apoptosis in differentiated thyroid cancer (DTC) cells in a synergistic manner. Three human DTC cell lines were used. NF-κB inhibition was achieved by using a NF-κB inhibitor (Bay 11-7082) or by p65 siRNA transfection. Methyl-thiazolyl-tetrazolium assay was performed for cell viability assessment. DNA-binding assay, luciferase reporter assay, and Western blot were adopted to determine function and expression changes of NF-κB. Then NF-κB regulated anti-apoptotic factors XIAP, cIAP1, and Bcl-xL were measured. Apoptosis was analyzed by Western blot for caspase 3 and PARP, and by flow cytometry as well. An iodide uptake assay was performed to determine whether NF-κB inhibition could influence radioactive iodide uptake. The methyl-thiazolyl-tetrazolium assay showed significant decrease of viable cells by combination therapy than by mono-therapies. The DNA-binding assay and luciferase reporter assay showed enhanced NF-κB function and reporter gene activities due to ¹³¹I, yet significant suppression was achieved by NF-κB inhibition. Western blot proved ¹³¹I could increase nuclear NF-κB concentration, while NF-κB inhibition reduced NF-κB concentration. Western blot also demonstrated significant up-regulation of XIAP, cIAP1, and Bcl-xL after ¹³¹I therapy. And inhibition of NF-κB could significantly down-regulate these factors. Finally, synergism induced by combined therapy was displayed by significant enhancements of cleaved caspase 3 and PARP from Western blot, and of Annexin V positively staining from flow cytometry. The iodine uptake assay did not show significant changes when NF-κB was inhibited. We demonstrated that ¹³¹I could induce NF-κB activation, which would attenuate ¹³¹I efficacy in DTC cells. NF-κB inhibition by Bay 11-7082 or by p65 siRNA transfection was effective in suppressing NF-κB regulated anti-apoptotic changes and in combined regimen apoptosis was achieved synergistically.