A new formulation of Bologna-type sausage enriched in ω-3 polyunsaturated fatty acids (PUFA) (8.75% linseed oil) was developed, using a lyophilized aqueous-ethanolic extract of Melissa officinalis. A comparison with the effectiveness of butylhydroxy anisole (BHA) synthetic antioxidant to decrease the oxidation of PUFAs was performed.The formulation increased the ω-3 PUFAs content, especially α-linolenic acid, decreasing significantly the ω-6/ω-3 ratio from 17.3 to 1.9, and also the Atherogenic Index and Thrombogenic Index (0.38-0.31 and 1.03-0.54, respectively).Modified sausages with BHA and Melissa extract showed significantly lower peroxides value (2.62 and 6.11meqO 2 /kg) and thiobarbituric acid value (0.26 and 0.27mg malondialdehyde/kg) and higher antioxidant capacity (hydrophilic fraction ABTS: 0.45 and 0.74meq Trolox/g product; lipofilic fraction ABTS: 0.44 and 0.37meq Trolox/g product) than those without these ingredients (16.49meq O 2 /kg, 2.08 mg malondialdehyde /kg, 0.26 and 0.27meq Trolox/g product, respectively). Sensorial tests showed that acceptability of the new formulations was similar to control products.

Neuroblastoma has a very diverse clinical behaviour: from spontaneous regression to a very aggressive malignant progression and resistance to chemotherapy. This heterogeneous clinical behaviour might be due to the existence of Cancer Stem Cells (CSC), a subpopulation within the tumor with stem-like cell properties: a significant proliferation capacity, a unique self-renewal capacity, and therefore, a higher ability to form new tumors. We enriched the CSC-like cell population content of two commercial neuroblastoma cell lines by the use of conditioned cell culture media for neurospheres, and compared genomic gains and losses and genome expression by array-CGH and microarray analysis, respectively (in CSC-like versus standard tumor cells culture). Despite the array-CGH did not show significant differences between standard and CSC-like in both analyzed cell lines, the microarray expression analysis highlighted some of the most relevant biological processes and molecular functions that might be responsible for the CSC-like phenotype. Some signalling pathways detected seem to be involved in self-renewal of normal tissues (Wnt, Notch, Hh and TGF-β) and contribute to CSC phenotype. We focused on the aberrant activation of TGF-β and Hh signalling pathways, confirming the inhibition of repressors of TGF-β pathway, as SMAD6 and SMAD7 by RT-qPCR. The analysis of the Sonic Hedgehog pathway showed overexpression of PTCH1, GLI1 and SMO. We found overexpression of CD133 and CD15 in SIMA neurospheres, confirming that this cell line was particularly enriched in stem-like cells. This work shows a cross-talk among different pathways in neuroblastoma and its importance in CSC-like cells.

In this work, we aimed at analyzing genome-wide gene expression and DNA gains and losses in neuroblastoma cell lines and in neurospheres containing stem-like cells. SKNDZ and SIMA neuroblastoma cell lines were grown in DMEM cell culture medium, and in DMEM-F12 selective serum free medium (with EGF, bFGF and B27; to induce the neurosphere-forming phenotype). After RNA and genomic DNA extractions from the 4 cell lines (2 standard, 2 neurosphere-forming cell lines), expression microarrays and array-CGH analyses were performed (Aligent Technologies). Array-CGH did not show any significant differences between standard and neurosphere-forming cell lines, both in SKNDZ and in SIMA. Microarray expression analysis demonstrated a total of 425 upregulated and 170 downregulated genes in neurosphere-forming cell lines. The differentially expressed genes were classified using the PANTHER classification system (www.pantherdb.org). As a result, apoptosis, cell adhesion, cell communication, cell cycle, and immune system processes appeared upregulated and downregulated in neurospheres. Some of those genes participate in pathways related with cancer (Table 1). In conclusion, the stem-like phenotype does not seem to be linked to anatomic changes at the level of deletions/gains of DNA, but to changes in expression of genes, like those of the TGF-beta, Notch and Sonic Hedgehog pathways. Those pathways, specially TGF-beta, widely described as an important therapeutic target against cancer, should be further studied to determine their real implication in the neurosphere-formation process in neuroblastoma, and therefore, as candidate targets for the treatment of neuroblastoma stem-like cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 413. doi:1538-7445.AM2012-413

Neuroblastoma, the most common extracranial malignant solid tumor in childhood has a very diverse clinical behaviour: from spontaneous regression to a very aggressive malignant progression and resistance to chemotherapy. This heterogeneous clinical behaviour might be due to molecular differences in tumor cell subpopulations. The cancer stem cells (CSC) model proposes the existence of a subpopulation within the tumor with stem-like cell properties: a significant proliferation capacity, a unique self-renewal capacity, and therefore, a higher ability to form new tumors. We enriched the stem-like cell population content of two commercial neuroblastoma cell lines (SKNDZ and SIMA) by the use of conditioned cell culture media for neurospheres, and compared genome expression and genomic gains and losses appearing in neurospheres versus the alterations appearing in standard tumor cells, by microarray analysis and array-CGH, respectively. Array-CGH did not show any significant differences between standard and neurosphere-forming cell lines, both in SKNDZ and in SIMA. The microarray expression analysis highlighted some of the most relevant biological processes and molecular functions that might be responsible for the stem-cell like phenotype. As expected, some signalling pathways detected were involved in self-renewal of normal tissues (Wnt, Notch, Hedgehog and TGF-β) and seem to contribute to CSC phenotype when deregulated. Among them, we focused on the aberrant activation of Hedgehog and TGF-β signalling pathways. We confirmed the inhibition of repressors of TGF-β pathway, as SMAD6 and SMAD7 by quantitative PCR. The analysis of the Sonic Hedgehog pathway showed overexpression of PTCH1, GLI1 and SMO. We analyzed two CSC surface markers, and found overexpression of CD133 and CD15 in SIMA neurospheres, confirming that this cell line was particularly enriched in stem-like cells. This work shows a cross-talk of different pathways in neuroblastoma, and the importance of it in stem-like cells, as confirmed by the overexpression observed in JAG1, one of the targets of TGF-β and also the main activator of Notch signalling pathway. Further analysis based on this work could identify possible new targets for molecular CSC therapies against neuroblastoma, highlighting the importance of redirecting current cancer treatments towards CSC to achieve total elimination of tumor cell population and improve treatment effectiveness. Citation Format: Raquel Ordoñez, Gabriel Gallo, Soledad Martínez, Sheila Legarra, Noémie Pata-Merci, Justine Guegan, Giselle Danglot, Xing Fan, Juan A. Rey, Alain Bernheim, Javier S. Castresana. Genome-wide microarray expression and genomic alteration by array-CGH analysis in neuroblastoma stem-like cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3051. doi:10.1158/1538-7445.AM2014-3051