Drawn from the cell culture medium, suggestive of a longterm effect on the population of stem-like cells. These observations are consistent with the persistent 115103-85-0 apoptosis detected in the post-treatment phase of cells which received RA/MG132 and with the enhanced levels of cleaved-caspase 3, long-term loss of expression of stemness markers and by the high percentage of dead cells detected by flow cytometry. To determine whether this marked cell death was related with secretion of self-renewing growth factors, or alternatively to an inherent mechanism already activated in cells, cultures were maintained in conditioned medium after drug withdrawal. However, even under these conditions, apoptosis persisted and the expression levels of Oct4 and Nestin were comparable to cells which were grown in normal medium after drug removal. Flow cytometry revealed a reduction of Nestinexpressing live cells after completion of combined treatment. Expression of Oct4, CD34 and Nanog was also diminished in live cells after the combined treatment. However, no differences in the expression levels of these three markers were observed among the different treatments. Therapy for high-risk patients includes the differentiating agent RA; however more than 50 of patients relapse, which could be due, at least in part, to the generation of resistance to retinoid. Thus, more information about the effects of RA is needed to improve the retinoid component of therapy for patients with minimal residual disease and to develop synergistic combination therapies which include RA. In the present study, we analyzed the effects of RA combined with proteasome inhibition in SK-N-BE neuroblastoma cells. This combined treatment caused apoptosis in a dose-dependent manner together with growth arrest and differentiation. The dose of the proteasome inhibitor MG132 that we employed to obtain apoptosis rates of about 50 was relatively high when compared to other studies in which Bortezomib was used on neuroblastoma cell lines. Peptide boronic acid proteasome inhibitors which include Bortezomib are more potent than their peptide aldehyde analogues such as MG132. A Thr-Pro-Pro-Thr-NH2 biological activity recent study has suggested that the sensitivity to proteasome inhibition is associated with the status of p53. However, apoptosis triggered by proteaso
