Prolonged activation of NF-kappaB is involved in the pathogenesis of chronic inflammatory diseases and associated cancers. NF-kappaB activation is considered to be a main mechanism opposing TNFalpha-induced apoptosis. We investigated whether inhibition of NF-kappaB could sensitize tumor and endothelial cells to TNFalpha-induced apoptosis. As such, we developed a novel H1 RNA polymerase III promoter driven adenoviral vector to express an RNA aptamer, Ad-A-p50, which selectively inhibits NF-kappaB activation in the nucleus. This event sensitizes human lung adenocarcinoma cells (A549) and human endothelial cells (HUVEC) to TNFalpha-induced apoptosis through the multiple pathways regulated by NF-kappaB, including Bcl-XL, HIF-1alpha, and VEGF. Our findings also suggest a new mechanism of HIF-1alpha regulation by NF-kappaB in the normoxic environment. RNA aptamer inhibition of NF-kappaB offers exciting opportunities for sensitizing inflammatory and tumor cells to TNFalpha-induced apoptosis.