Doxorubicin and α-Mangostin oppositely affect luminal breast cancer cell stemness evaluated by a new retinaldehyde-dependent ALDH assay in MCF-7 tumor spheroids

According to cancer stem cell theory, only a limited number of self-renewing and cloning cells are responsible for tumor relapse after a period of remittance. The aim of the present study was to investigate the effects of Doxorubicin and α-Mangostin, two antiproliferative drugs, on both tumor bulk and stem cells in multicellular tumor spheroids originated from the luminal MCF-7 breast cancer cell line. A new and original fluorimetric assay was used to selectively measure the activity of the retinaldehyde-dependent isoenzymes of aldehyde dehydrogenase (RALDH), which are markers of a subpopulation of breast cancer stem cells. The administration of 5 μg/ml (12.2 μM) α-Mangostin for 48 h provoked: i) a marked disaggregation of the spheroids, leading to a doubling of their volume (p < 0.01), ii) a 40 % decrease in cell viability (p < 0.01), evaluated by the acid phosphatase assay, and iii) a reduction by more than 90 % of RALDH activity. By contrast, Doxorubicin given for 48 h in the range of 0.1–40 μM did not significantly reduce cell viability and caused only a modest modification of the spheroid morphology. Moreover, 40 μM Doxorubicin increased RALDH activity 2.5-fold compared to the untreated sample. When the two drugs were administered together using 5 μg/ml α-Mangostin, the IC50 of Doxorubicin referred to cell viability decreased six-fold and the RALDH activity was further reduced. In conclusion, the combined administration of Doxorubicin and α-Mangostin provoked a significant cytotoxicity and a remarkable inhibition of RALDH activity in MCF-7 tumor spheroids, suggesting that these drugs could be effective in reducing cell stemness in luminal breast cancer

NOTCH1: A Novel Player in the Molecular Crosstalk Underlying Articular Chondrocyte Protection by Oleuropein and Hydroxytyrosol

Osteoarthritis (OA) is the most common joint disease, but no effective and safe disease-modifying treatment is available. Risk factors such as age, sex, genetics, injuries and obesity can concur to the onset of the disease, variably triggering the loss of maturational arrest of chondrocytes further sustained by oxidative stress, inflammation and catabolism. Different types of nutraceuticals have been studied for their anti-oxidative and anti-inflammatory properties. Olive-derived polyphenols draw particular interest due to their ability to dampen the activation of pivotal signaling pathways in OA. Our study aims to investigate the effects of oleuropein (OE) and hydroxytyrosol (HT) in in vitro OA models and elucidate their possible effects on NOTCH1, a novel therapeutic target for OA. Chondrocytes were cultured and exposed to lipopolysaccharide (LPS). Detailed analysis was carried out about the OE/HT mitigating effects on the release of ROS (DCHF-DA), the increased gene expression of catabolic and inflammatory markers (real time RT-PCR), the release of MMP-13 (ELISA and Western blot) and the activation of underlying signaling pathways (Western blot). Our findings show that HT/OE efficiently attenuates LPS-induced effects by firstly reducing the activation of JNK and of the NOTCH1 pathway downstream. In conclusion, our study provides molecular bases supporting the dietary supplementation of olive-derived polyphenols to revert/delay the progression of OA

PRX2 and PRX25, peroxidases regulated by COG1, are involved in seed longevity in Arabidopsis

[EN] Permeability is a crucial trait that affects seed longevity and is regulated by different polymers including proanthocyanidins, suberin, cutin and lignin located in the seed coat. By testing mutants in suberin transport and biosynthesis, we demonstrate the importance of this biopolymer to cope with seed deterioration. Transcriptomic analysis of cog1-2D, a gain-of-function mutant with increased seed longevity, revealed the upregulation of several peroxidase genes. Reverse genetics analysing seed longevity uncovered redundancy within the seed coat peroxidase gene family; however, after controlled deterioration treatment, seeds from the prx2 prx25 double and prx2 prx25 prx71 triple mutant plants presented lower germination than wild-type plants. Transmission electron microscopy analysis of the seed coat of these mutants showed a thinner palisade layer, but no changes were observed in proanthocyanidin accumulation or in the cuticle layer. Spectrophotometric quantification of acetyl bromide-soluble lignin components indicated changes in the amount of total polyphenolics derived from suberin and/or lignin in the mutant seeds. Finally, the increased seed coat permeability to tetrazolium salts observed in the prx2 prx25 and prx2 prx25 prx71 mutant lines suggested that the lower permeability of the seed coats caused by altered polyphenolics is likely to be the main reason explaining their reduced seed longevityRenard, J.; Martínez-Almonacid, I.; Sonntag, A.; Molina, I.; Moya-Cuevas, J.; Bissoli, G.; Muñoz-Bertomeu, J.... (2020). PRX2 and PRX25, peroxidases regulated by COG1, are involved in seed longevity in Arabidopsis. Plant Cell & Environment. 43(2):315-326. https://doi.org/10.1111/pce.13656S315326432Almagro, L., Gómez Ros, L. V., Belchi-Navarro, S., Bru, R., Ros Barceló, A., & Pedreño, M. A. (2008). Class III peroxidases in plant defence reactions. 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C., & Koornneef, M. (2004). Genetic differences in seed longevity of various Arabidopsis mutants. Physiologia Plantarum, 121(3), 448-461. doi:10.1111/j.0031-9317.2004.00339.xCosio, C., & Dunand, C. (2009). Specific functions of individual class III peroxidase genes. Journal of Experimental Botany, 60(2), 391-408. doi:10.1093/jxb/ern318Czechowski, T., Stitt, M., Altmann, T., Udvardi, M. K., & Scheible, W.-R. (2005). Genome-Wide Identification and Testing of Superior Reference Genes for Transcript Normalization in Arabidopsis. Plant Physiology, 139(1), 5-17. doi:10.1104/pp.105.063743Debeaujon, I., Léon-Kloosterziel, K. M., & Koornneef, M. (2000). Influence of the Testa on Seed Dormancy, Germination, and Longevity in Arabidopsis. Plant Physiology, 122(2), 403-414. doi:10.1104/pp.122.2.403Duroux, L., & Welinder, K. G. (2003). The Peroxidase Gene Family in Plants: A Phylogenetic Overview. Journal of Molecular Evolution, 57(4), 397-407. doi:10.1007/s00239-003-2489-3Fedi, F., O’Neill, C. 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A Tomato Peroxidase Involved in the Synthesis of Lignin and Suberin. Plant Physiology, 122(4), 1119-1128. doi:10.1104/pp.122.4.1119Rains, M. K., Gardiyehewa de Silva, N. D., & Molina, I. (2017). Reconstructing the suberin pathway in poplar by chemical and transcriptomic analysis of bark tissues. Tree Physiology, 38(3), 340-361. doi:10.1093/treephys/tpx060Russell, W. R., Burkitt, M. J., Scobbie, L., & Chesson, A. (2005). EPR Investigation into the Effects of Substrate Structure on Peroxidase-Catalyzed Phenylpropanoid Oxidation. Biomacromolecules, 7(1), 268-273. doi:10.1021/bm050636oSano, N., Rajjou, L., North, H. M., Debeaujon, I., Marion-Poll, A., & Seo, M. (2015). Staying Alive: Molecular Aspects of Seed Longevity. Plant and Cell Physiology, 57(4), 660-674. doi:10.1093/pcp/pcv186Shigeto, J., Itoh, Y., Hirao, S., Ohira, K., Fujita, K., & Tsutsumi, Y. (2015). Simultaneously disrupting AtPrx2 , AtPrx25 and AtPrx71 alters lignin content and structure in Arabidopsis stem. 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Identification of novel seed longevity genes related to oxidative stress and seed coat by genome wide association studies and reverse genetics

[EN] Seed longevity is a polygenic trait of relevance for agriculture and for understanding the effect of environment on the ageing of biological systems. In order to identify novel longevity genes, we have phenotyped the natural variation of 270 ecotypes of the model plant,Arabidopsis thaliana, for natural ageing and for three accelerated ageing methods. Genome-wide analysis, using publicly available single-nucleotide polymorphisms (SNPs) data sets, identified multiple genomic regions associated with variation in seed longevity. Reverse genetics of 20 candidate genes in Columbia ecotype resulted in seven genes positive for seed longevity (PSAD1,SSLEA,SSTPR,DHAR1,CYP86A8,MYB47andSPCH) and five negative ones (RBOHD,RBOHE,RBOHF,KNAT7andSEP3). In this uniform genetic background, natural and accelerated ageing methods provided similar results for seed-longevity in knock-out mutants. The NADPH oxidases (RBOHs), the dehydroascorbate reductase (DHAR1) and the photosystem I subunit (PSAD1) highlight the important role of oxidative stress on seed ageing. The cytochrome P-450 hydroxylase, CYP86A8, and the transcription factors, MYB47, KNAT7 and SEP3, support the protecting role of the seed coat during seed ageing.Ministerio de Ciencia, Innovacion y Universidades, Grant/Award Number: BIO2017-88898-PRenard, J.; Niñoles Rodenes, R.; Martínez-Almonacid, I.; Gayubas, B.; Mateos-Fernández, R.; Bissoli, G.; Bueso Rodenas, E.... (2020). Identification of novel seed longevity genes related to oxidative stress and seed coat by genome wide association studies and reverse genetics. Plant Cell & Environment. 43(10):2523-2539. https://doi.org/10.1111/pce.13822S25232539431

Doxorubicin and \u3b1-Mangostin oppositely affect luminal breast cancer cell stemness evaluated by a new retinaldehyde-dependent ALDH assay in MCF-7 tumor spheroids

According to cancer stem cell theory, only a limited number of self-renewing and cloning cells are responsible for tumor relapse after a period of remittance. The aim of the present study was to investigate the effects of Doxorubicin and \u3b1-Mangostin, two antiproliferative drugs, on both tumor bulk and stem cells in multicellular tumor spheroids originated from the luminal MCF-7 breast cancer cell line. A new and original fluorimetric assay was used to selectively measure the activity of the retinaldehyde-dependent isoenzymes of aldehyde dehydrogenase (RALDH), which are markers of a subpopulation of breast cancer stem cells. The administration of 5 \u3bcg/ml (12.2 \u3bcM) \u3b1-Mangostin for 48 h provoked: i) a marked disaggregation of the spheroids, leading to a doubling of their volume (p &lt; 0.01), ii) a 40 % decrease in cell viability (p &lt; 0.01), evaluated by the acid phosphatase assay, and iii) a reduction by more than 90 % of RALDH activity. By contrast, Doxorubicin given for 48 h in the range of 0.1\u201340 \u3bcM did not significantly reduce cell viability and caused only a modest modification of the spheroid morphology. Moreover, 40 \u3bcM Doxorubicin increased RALDH activity 2.5-fold compared to the untreated sample. When the two drugs were administered together using 5 \u3bcg/ml \u3b1-Mangostin, the IC50 of Doxorubicin referred to cell viability decreased six-fold and the RALDH activity was further reduced. In conclusion, the combined administration of Doxorubicin and \u3b1-Mangostin provoked a significant cytotoxicity and a remarkable inhibition of RALDH activity in MCF-7 tumor spheroids, suggesting that these drugs could be effective in reducing cell stemness in luminal breast cancer