Quassinoids: From traditional drugs to new cancer therapeutics.

Quassinoids are a group of compounds extracted from plants of the Simaroubaceae family, which have been used for many years in folk medicine. These molecules gained notoriety after the initial discovery of the anti-leukemic activity of one member, bruceantin, in 1975. Currently over 150 quassinoids have been isolated and classified based on their chemical structures and biological properties investigated in vitro and in vivo. Many molecules display a wide range of inhibitory effects, including anti-inflammatory, anti-viral, anti-malarial and anti-proliferative effects on various tumor cell types. Although often the exact mechanism of action of the single agents remains unclear, some agents have been shown to affect protein synthesis in general, or specifically HIF-1α and MYC, membrane polarization and the apoptotic machinery. Considering that future research into chemical modifications is likely to generate more active and less toxic derivatives of natural quassinoids, this family represents a powerful source of promising small molecules targeting key prosurvival signaling pathways relevant for diverse pathologies. Here, we review available knowledge of functionality and possible applications of quassinoids and quassinoid derivatives, spanning traditional use to the potential impact on modern medicine as cancer therapeutics.

Bone morphogenetic protein-7 is a MYC target with prosurvival functions in childhood medulloblastoma.

Medulloblastoma (MB) is the most common malignant brain tumor in children. It is known that overexpression and/or amplification of the MYC oncogene is associated with poor clinical outcome, but the molecular mechanisms and the MYC downstream effectors in MB remain still elusive. Besides contributing to elucidate how progression of MB takes place, most importantly, the identification of novel MYC-target genes will suggest novel candidates for targeted therapy in MB. A group of 209 MYC-responsive genes was obtained from a complementary DNA microarray analysis of a MB-derived cell line, following MYC overexpression and silencing. Among the MYC-responsive genes, we identified the members of the bone morphogenetic protein (BMP) signaling pathway, which have a crucial role during the development of the cerebellum. In particular, the gene BMP7 was identified as a direct target of MYC. A positive correlation between MYC and BMP7 expression was documented by analyzing two distinct sets of primary MB samples. Functional studies in vitro using a small-molecule inhibitor of the BMP/SMAD signaling pathway reproduced the effect of the small interfering RNA-mediated silencing of BMP7. Both approaches led to a block of proliferation in a panel of MB cells and to inhibition of SMAD phosphorylation. Altogether, our findings indicate that high MYC levels drive BMP7 overexpression, promoting cell survival in MB cells. This observation suggests the potential relevance of targeting the BMP/SMAD pathway as a novel therapeutic approach for the treatment of childhood MB.

Peptide analogues of a T-cell epitope of ricin toxin A-chain prevent agonist-mediated human T-cell response.

The clinical efficacy of immunotoxins (IT) containing ricin toxin A-chain (RTA) can be drastically reduced by anti-toxin-neutralizing antibodies developed by patients. Strategies aimed at epitope-specific modulation of the immune response must be therefore set up to broaden the clinical applicability of RTA-based IT. Prevention or reduction of humoral immune responses against RTA could be achieved by peptide-based down-modulating strategies. Peptide analogues were investigated as candidate antagonist altered peptide ligands (APL) considering the sequence of a previously identified dominant T-cell epitope of RTA (i.e. I175-E185) presented in the context of the HLA-DRB1*03011 allele. Alanine-substituted peptides provided information on the role of individual residues of the wild-type peptide and allowed to identify one antagonist APL corresponding to the double-mutant peptide E177A/A178D. The analogue E177A/A178D not only prevented the agonist from stimulating anti-RTA human T-cell clones but also failed to induce down-regulation of surface-expressed TCR, thus suggesting its possible use for in vivo immune modulation of anti-RTA responses.

A dominant linear B-cell epitope of ricin A-chain is the target of a neutralizing antibody response in Hodgkin's lymphoma patients treated with an anti-CD25 immunotoxin.

Hodgkin's lymphoma patients treated with an anti-CD25 Ricin toxin A-chain (RTA)-based Immunotoxin (RFT5.dgA) develop an immune response against the toxic moiety of the immunoconjugate. The anti-RTA antibody response of 15 patients showing different clinical features and receiving different total amounts of RFT5.dgA was therefore studied in detail, considering antibody titre, IgG and IgM content, average binding efficacy and ability to inhibit in vitro the cytotoxicity of a RTA-based Immunotoxin. No correlations were found between these parameters and the clinical features of the patients or the total amount of Immunotoxin administered. However, using a peptide scan approach we have identified a continuous epitope recognized by all patients studied, located within the stretch L161-I175 of the RTA primary sequence, close to a previously identified T-cell epitope. The ability of anti-L161-I175 antibodies to recognize folded RTA and to affect the biological activity of RTA by inhibiting RTA-IT cytotoxicity in vitro revealed that they may exert an important role in IT neutralization in vivo. Discovery of RTA immunodominant epitopes which are the target of anti-RTA immune response may lead to the development of immunomodulating strategies and to more successful treatment schedules.

Identification of ricin A-chain HLA class II-restricted epitopes by human T-cell clones.

The identification of ricin toxin A-chain (RTA) epitopes and the molecular context in which they are recognized will allow strategies to be devised that prevent/suppress an anti-RTA immune response in patients treated with RTA-based immunotoxins. RTA-specific human T-cell lines and T-cell clones were produced by in vitro priming of PBMC. The T-cell clones used a limited set of Vbeta chains (Vbeta1, Vbeta2 and Vbeta8) to recognize RTA epitopes. The use of RTA deletion mutants demonstrated that T-cell lines and T-cell clones from three out of four donors responded to RTA epitopes within the domain D124-Q223, whereas one donor recognized the region I1-D124. The response to RTA peptides of T-cell lines and T-cell clones from two donors allowed the identification of immunogenic segments (D124-G140 and L161-T190) recognized in the context of different HLA-DRB1 alleles (HLA-DRB1*0801, and HLA-DRB1*11011 and B1*03011, respectively). The response to L161-T190 was investigated in greater detail. We found that the HLA-DRB1*03011 allele presents a minimal epitope represented by the sequence I175-Y183 of RTA, whereas the HLA-DRB1*11011 allele presents the minimal epitope M174-I184. RTA peptides and an I175A RTA point mutant allowed us to identify I175 as a crucial residue for the epitope(s) recognized by the two HLA-DRB1 alleles. Failure of T-cell clones to recognize ribosome inactivating proteins (RIPs) showing sequences similar but not identical to RTA further confirmed the role of I175 as a key residue for the epitope recognized in the context of HLA-DRB1*11011/03011 alleles.