Thus, eradicating tumors may be difficult because conventional treatments target the bulk of tumor cells rather than these tumor-initiating stem cells which are chemoresistant. The sidepopulation assay is widely used for the identification and isolation of stem-like cells from cancers based on their capacity to exclude dyes such as Hoechst 33342. To determine whether the RA/MG132 combination alters the population of stem-like cells of neuroblastomas, we analysed the expression of stem cell-related markers such as Oct4, Nanog and Sox2 which are key regulators of embryonic stem cell maintenance and are overexpressed in different cancers, including neuroblastoma. The neural progenitor markers Nestin and CD34 are also expressed in neuroblastoma cells. The exact role of these stem cellrelated genes in tumors is not completely clear, but Nanog, Oct4 and Nestin have been associated with a more immature and aggressive cell phenotype. In our studies, protein levels of Oct4, Sox2, and Nanog were significantly reduced by RA/MG132 combined treatment. Remarkably, this reduction of stem cell markers persisted during the 5 days after treatment cessation. LY2157299 TGF-beta inhibitor moreover, the proportion of live cells expressing Nestin and Oct4 was significantly lower in cells receiving RA/MG132 than in control cultures or those treated with only one of the compounds. This effect was accompanied by persistent apoptosis and differentiation of those cells that escaped apoptosis, suggesting that RA/MG132 may be beneficial to prevent neuroblastoma relapse. The standard treatment for high-risk neuroblastoma patients includes RA. The objective of this approach is eradication of minimal residual disease which is present in over half of children who have achieved complete remission by imaging criteria. However, even with this intensive treatment, many children relapse and eventually die from disease progression. Our data reveal the novel observation that proteasome inhibition administered in combination with RA induces apoptosis in stem-cell like cells of neuroblastoma cell lines. The combined effects of RA/MG132 were more potent at reducing the stem-like cell population than either compound alone and moreover, impaired their capacity to form neurospheres. Therefore, we predict that this combined treatment might also have a positive impact in vivo in animal models. In human acute myeloid leukemia cells, bortezomib also sensitizes to RA-induced differentiation. However, our results also show increased apoptosis, suggesting that the molecular targets between these two diseases might be different, such as the activation of the JNK pathway, or on whether bortezomib is given after or concomitantly with RA. Since cancer stem cells 
are frequently resistant to conventional therapy and are responsible for relapse, our results suggest that dual therapy might be beneficial for improving the outcome of patients with high-risk neuroblastoma. RA is the current standard treatment in the control of minimal residual disease in high-risk neuroblastoma patients and Bortezomib is already approved by EMA/FDA. Development of therapies for pediatric cancers is complicated by the rarity of these diseases with respect to the total population and the fact that only a limited number of drugs can be tested. Hence, drug combination therapies, particularly with drugs that are already approved, may play a key role in future neuroblastoma treatment strategies. Small disulfide-rich SP600125 129-56-6 peptides from plants and animals have diverse structures and bioactivities, and many have potential therapeutic applications. The Cucurbitaceae plant family is a rich source of bioactive peptides with more than 60 disulfide-rich peptides isolated from over 10 species. One species that has been of particular interest is Momordica charantia Linn., a tropical and subtropical vine, which is widely grown as a vegetable. It is commonly known as bitter gourd or bitter melon because the fruit is among the most bitter of all fruits. The roots, vines and seeds of M. charantia are used in traditional Chinese medicines. Several serine protease inhibitors have been isolated and characterized from the seeds. These inhibitors are classified as squash trypsin inhibitors and are small disulfide-rich peptides containing three-disulfide bonds. Members of this family share the characteristic feature of an inhibitor cystine knot motif, in which an embedded ring, formed by the CysI-CysIV, CysII-CysV disulfide bonds and their connecting peptide backbone segments, is penetrated by the CysIII-CysVI disulfide bond.