Chemosensitization by diverging modulation by short-term and long-term TNF-α action on ABCB1 expression and NF-κB signaling in colon cancer

Multidrug resistance (MDR) is a major cause for cancer chemotherapy failure. Among the numerous strategies to overcome persistent action of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha) permits down-regulation of MDR-associated genes, including ATP-binding cassette, subfamily B 1 gene (ABCB1). A key regulator of ABCB1 expression is the transcription factor nuclear factor light chain enhancer (NF-kappa B)/p65. We analyzed diverging short- and long-term effects of TNF-alpha regarding modulation of NF-kappa B/p65 signaling and ABCB1 expression in colon cancer cells. Highly resistant ABCB1 overexpressing human HCT15 colorectal carcinoma cells were subjected to short- (30-120 min) or long-term (24-96 h) TNF-alpha treat: ment. TNF-alpha mediated modulation of ABCB1 expression was analyzed by real-time RT-PCR and western blot analysis. The TNF-mediated chemosensitization was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay. The involvement of TNF receptors and of NF-kappa B/p65 signaling was analyzed by western blot analysis, ABCB1 promoter analysis and electrophoretic mobility shift assay (EMSA). The study revealed, that long-term, but not short-term TNF-alpha treatment leads to TNF-receptor 1 (TNFR1) mediated downregulation of ABCB1 resulting in sensitization towards drug treatment. It dampens NF-kappa B/p65 activation and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (I kappa B alpha) resynthesis, associated with reduced nuclear accumulation of NF-kappa B/p65 and reduced binding to its consensus sequence in the ABCB1 promoter. The study reveals the diverging effects of short- or long-term TNF-alpha action and provides novel insights on downregulation of ABCB1 expression by TNF-mediated repression of NF-kappa B signaling.