Retinoic acid therapy is nowadays an important component of treatment for residual disease of stage IV neuroblastoma after multimodal therapy. Nevertheless, arising resistance and treatment toxicity could represent relevant limiting factors. In the present study, we show that retinoic acid enhances the cytostatic and apoptogenic properties of the novel adamantyl retinoid ST1926 in a panel of neuroblastoma cells with different p53 status and caspase 8 expression, resulting in synergistic effects as assessed by Combination Index and Isobologram analysis. Under conditions where the two drugs alone produced no toxic effects, their combination resulted in enhanced G2-M arrest and sub-G1 population as shown by BrdU pulse-chase and labeling experiments. PARP cleavage, caspase 3, 8 and 9 activation and modulation of DR4 and FAS were indicative of enhanced apoptosis triggered by the co-incubation of the two drugs whereas neither ST1926-mediated genotoxic damage nor ATRA-differentiating effects were affected by the combined treatment. Caspase-3 and 8-mediated apoptosis appeared to play an important role in the drugs synergism. In fact, the addition of a pan-caspase inhibitor ZVAD-FMK reverted this effect in SK-N-DZ cells, and synergism was confined to limited drugs doses in HTLA cells not expressing caspase-8. Although not modulated, p53 appeared to enhance cells responsiveness to retinoid/ATRA combination. In vivo studies in the most sensitive neuroblastoma model SK-N-DZ, confirmed enhanced activity of the drugs combination vs single treatments. The study provides important lines of evidence that such a drugs combination could represent a less toxic and more effective approach for maintenance treatment in children with neuroblastoma.
Figure 1SMGrowth Inhibition curves of ST1926 and ATRA (24 h treatment) in the panel of neuroblastoma cell lines by 7-day SRB assay. The data are a mean of three independent experiments (±SD). (PPT 110 kb)
Figure 2SMCell morphology study on SK-N-DZ cells treated with either single drugs (ATRA 2.5 μM or ST1926 0.2 μM) or their combination for up to 13 days. The phase contrast images shown, were acquired on the 6th day of treatment with a digital camera (Canon, Power Shot G5) fixed on a Olympus reverse microscope at 10x magnification. (PPT 3497 kb)
Figure 3SMAlcaline Comet Assay (ACA) on SK-N-DZ cells treated with ST1926 0.2 μM, ATRA 2.5 μM or the combination of the two drugs, for 24 h (white bars). A second set of drug-treated samples was left for further 24 h in drug-free medium (grey bars). Vehicle-treated cells (ctr) and H2O2-treated cells (50 μM, 5 min on ice) represented the negative and positive controls, respectively. The data are representative of two independent experiments run in duplicate. Errors (±SD) are reported. (PPT 684 kb)