The apoptosis-inducing effect of gastrin on colorectal cancer cells relates to an increased IEX-1 expression mediating NF-κB inhibition

Addressing the puzzling role of amidated gastrin(17) ( G17) and the gastrin/ CCKB/ CCK2 receptor in colorectal carcinogenesis, we analysed potential candidate genes involved in G17-dependent NF-kappa B inhibition and apoptosis. The colorectal carcinoma cell line Colo320 over-expressing the wild-type CCK2 receptor ( Colo320wt) underwent G17-induced apoptosis along with suppressed NF-kappa B activation and decreased expression of the antiapoptotic NF-kappa B target genes cIAP1 and cIAP2, whereas G17 was without effect on Colo320 cells expressing a CCK2 receptor bearing a loss of function mutation ( Colo320mut). Gene microarray analysis revealed an elevated expression of the stress response gene IEX-1 in G17-treated Colo320wt but not Colo320mut cells. Quantitative real-time PCR and conventional RT-PCR confirmed this G17-dependent increase of IEX-1 expression in Colo320wt cells. If these cells were subjected to IEX-1 knockdown by small interfering RNA transfection, the apoptosis-inducing effect of G17 was abolished. Moreover, tumor necrosis factor alpha (TNF alpha)- or 5-FU-induced apoptosis that is greatly enhanced by G17 treatment in Colo320wt cells was prevented if IEX-1 expression was repressed. Under these conditions of blocked IEX-1 expression, the NF-kappa B activity remained unaffected by G17, in particular in Colo320wt cells co-treated with TNF alpha and also the suppressive effect of G17 on cIAP1 and cIAP2 expression was not observed anymore if IEX-1 expression was blocked. Conversely, IEX-1 overexpression in Colo320mut cells caused an increase of basal and TNF alpha- or 5-FU-induced apoptosis, an effect not further triggered by G17 treatment. Using a xenograft tumor model in severe combined immune deficiency mice, we could show that experimental systemic hypergastrinemia induced by the administration of omeprazole led to enhanced apoptosis as well as to a marked increase of IEX-1 expression in Colo320wt tumors, but not in Colo320mut tumors. These observations indicate that the proapoptotic effect of G17 on human colon cancer cells expressing the wild-type CCK2 receptor is mediated by IEX-1, which modulates NF-kappa B-dependent antiapoptotic protection and thereby exerts tumor-suppressive potential.