Significant decreases of interleukin-1α gene expression after application of the serotonin receptor antagonist ondansetron are found to correlate with antiproliferative properties in the acute lymphoblastic leukemia cell line REH

As recently reported in a previous article from our group [1], ondansetron has been found to exhibit antiproliferative properties in vitro on acute lymphoblastic leukemia (ALL) cells. Ondansetron is a serotonin (5-hydroxytryptamine, 5-HT) receptor-3 (5-HT 3 ) antagonist, which is widely used as an antiemetic agent in the prophylactic treatment of chemotherapy induced nausea and vomiting (CINV) in patients with ALL [2]. Moreover, in further articles from our group in Berlin [3,4], it has also been reported that the antiproliferative properties of ondansetron on ALL cells are associated with increases in the production of interferon- γ (IFN γ ) [3] and nitric oxides (NO) [4]. Since the release of IFN γ and NO is often related to the production of infl ammatory cytokines, it is of interest to analyze the eff ects of ondansetron on the expression of such cytokines in ALL cells, for example the case of interleukin-1 α (IL-1 α ). In all experiments performed, the well-known cell line REH was used; this was derived from a patient with ALL at fi rst relapse [5], and actually represents one of the best characterized B-cell precursor (BCP)-ALL cell lines, being considered one of the most representative cell lines for in vitro studies in childhood ALL [5,6]. In our study, REH cells in the logarithmic growth phase were cultured in 24-well culture plates at a cell concentration of 1 – 2 � 10 4 cells/mL (1 mL/well) in complete RPMI-1640 medium (Biochrom, Berlin, Germany), without addition of fetal calf serum and antibiotics. After the application of ondansetron, the cultured REH cells were further incubated for 72 h at 37 ° C in a humidifi ed atmosphere of 5% CO 2 in air, either in the absence or in the presence of added 10 μ M 5-HT. Dot-blot mRNA in situ hybridizations for the analysis of IL-1 α gene expression were performed using a highly specifi c gene probe for human IL-1 α , as previously described [7]. Total RNA was isolated from cell pellets with the aid of RNeasy O mini-columns (Qiagen GmbH, Hilden, Germany). All dot-blot hybridizations were carried out with the aid of a non-radioactive digoxigenin (DIG)-labeling and detection kit from Roche Diagnostics (Mannheim, Germany). Th e obtained mRNA dot-blot hybridization patterns were analyzed in a Molecular Imager GS-800 Calibrated Densitometer using Quantity One software (Bio-Rad Laboratories, Munich, Germany). Added 5-HT was purchased from Sigma-Aldrich, Taufkirchen (Germany), and ondansetron (Zofran O ) from Glaxo Wellcome GmbH, Munich (Germany). All the reported experiments were performed at least three times in double series of triplicates, and results were considered statistically signifi cant for p � 0.05 in the two-sided unpaired Student ’ s t -test. For correlation analysis, Pearson ’ s correlation coeffi cient (PCC) was used, which is known to vary between 1.0 (maximum) and 0.0 (minimum), thus indicating the “ maximum ” and “ minimum ” levels of correlation, respectively. After 72 h incubation in the standard culture conditions described above, reproducible amounts of IL-1 α mRNA were systematically detected and further quantifi ed in all the tested cell pellets, either in the absence or in the presence of 5 – 50 μ M ondansetron. Interestingly, the analyzed REH cells showed reproducible and signifi cant decreases in the expression of IL-1 α mRNA after 72 h co-incubation with 5 – 50 μ M ondansetron, either in the absence ( � 41% for 5 μ M ondansetron, p � 0.04949 * ; � 51% for 50 μ M ondansetron, p � 0.00006 * * ) or in the presence ( � 34% for 5 μ M ondansetron, p � 0.00952 * * ; � 43% for 50 μ M ondansetron, p � 0.00086 * * ) of 10 μ M exogenous 5-HT. In addition, just the presence of 10 μ M exogenous 5-HT alone was able to induce a reproducible reduction of IL-1 α gene expression ( � 28%, p � 0.00560 * * ), as well as in the presence of 5 μ M ( � 20%, p � 0.27186 � � 0.05; not signifi cant) or 50 μ M ondansetron ( � 17%, p � 0.02350 * ).