Insights into the control of taxane metabolism: Molecular, cellular, and metabolic changes induced by elicitation in Taxus baccata cell suspensions

More knowledge is needed about the molecular/cellular control of paclitaxel (PTX) production in Taxus spp. cell cultures. In this study, the yield of this anticancer agent in Taxus baccata cell suspensions was improved 11-fold after elicitation with coronatine (COR) compared to the untreated cells, and 18-fold when co-supplemented with methyl-β-cyclodextrins (β-CDs). In the dual treatment, the release of taxanes from the producer cells was greatly enhanced, with 81.6% of the total taxane content being found in the medium at the end of the experiment. The experimental conditions that caused the highest PTX production also induced its maximum excretion, and increased the expression of taxane biosynthetic genes, especially the flux-limiting BAPT and DBTNBT. The application of COR, which activates PTX biosynthesis, together with β - CDs, which form inclusion complexes with PTX and related taxanes, is evidently an efficient strategy for enhancing PTX production and release to the culture medium. Due to the recently described role of lipid droplets (LDs) in the trafficking and accumulation of hydrophobic taxanes in Taxus spp. cell cultures, the structure, number and taxane storage capacity of these organelles was also studied. In elicited cultures, the number of LDs increased and they mainly accumulated taxanes with a side chain, especially PTX. Thus, PTX constituted up to 50-70% of the total taxanes found in LDs throughout the experiment in the COR + β - CD-treated cultures. These results confirm that LDs can store taxanes and distribute them inside and outside cells. Keywords: Taxus baccata; cell cultures; coronatine; gene expression; lipid droplets; paclitaxel; taxane accumulation

Viability-reducing activity of Coryllus avellana L. extracts against human cancer cell lines

The increasing rate of cancer incidence has encouraged the search for novel natural sources of anticancer compounds. The presence of small quantities of taxol and taxanes in Corylus avellana L. has impelled new potential applications for this plant in the field of biomedicine. In the present work, the cell viability-reducing activity of stems and leaves from three different hazel trees was studied against three human-derived cancer cell lines (HeLa, HepG2 and MCF-7). Both leaf and stem extracts significantly reduced viability of the three cell lines either after maceration with methanol or using taxane extraction methods. Since maceration reduced cell viability to a greater extent than taxane extraction methods, we scaled up the maceration extraction process using a method for solid/liquid extraction (Zippertex technology). Methanol leaf extracts promoted a higher reduction in viability of all cell lines assayed than stem extracts. Fractionation of methanol leaf extracts using silica gel chromatography led to the purification and identification of two compounds by HPLC-MS and NMR: (3R,5R)-3,5-dihydroxy-1,7-bis(4-hydroxyphenyl) heptane 3-O-β-d-glucopyranoside and quercetin-3-O-rhamnoside. The isolated compounds decreased viability of HeLa and HepG2 cells to a greater extent than MCF-7 cells. Our results suggest a potential use of C. avellana extracts in the pharmacotherapy of cervical cancer and hepatocarcinoma and, to a lesser extent, breast cancer

The Role of Citizen Science in Promoting Ocean and Water Literacy in School Communities: The ProBleu Methodology

Human activities continue to degrade oceanic, coastal and inland waters. The generational change in the role of society in actively looking after the health of water resources can be achieved through the expansion of ocean and water literacy in schools. The Network of European Blue Schools established under the EU4Ocean Coalition for Ocean Literacy has improved ocean and water literacy; however, this Network needs to grow and be supported. Here, we present ProBleu, a recently funded EU project that will expand and support the Network, partly through the use of citizen science. The core of the proposed methodology is facilitating school activities related to ocean and water literacy through funding calls to sustain and enrich current school activities, and kick-start and support new activities. The outcomes of the project are anticipated to have widespread and long-term impacts across society, and oceanic, coastal and inland water environments

Associació Catalana Dones de la Mar: Un camí de visibilització, empoderament i xarxa entre dones

Charla organizada por ICM Young ResearchersPeer reviewe

Metabolic gene expression and centelloside production in elicited Centella asiatica hairy root cultures

The most economically important centellosides, madecassoside and asiaticoside, are produced in very low amounts in Centella asiatica (L.) Urban roots. With the aim of overcoming this limitation, in this work we compared the effect of different elicitors on centelloside production in hairy root cultures established by Agrobacterium rhizogenes infection. Additionally, elicitor-induced changes in the expression of key biosynthetic genes were studied to shed light on the regulation of the triterpene metabolic pathway. The elicitors tested were coronatine and methyl jasmonate, added separately or together, and salicylic acid. The content of the four main centellosides (asiaticoside, madecassoside, asiatic acid and madecassic acid) was determined by HPLC/DAD, and the expression level of genes after elicitation was analyzed by real-time quantitative polymerase chain reaction. The greatest increase in the production of centellosides, especially madecassoside, was achieved with coronatine, applied alone or with methyl jasmonate. This treatment also enhanced the expression of the target genes, particularly at the start of elicitation. By far the most highly expressed were those involved in oxidations, indicating that the tested elicitors did not act specifically on key genes in the centelloside biosynthetic pathway. These results support that hairy roots are a promising biotechnological platform for improved centelloside production and that this approach warrants further research.This work was partially funded by the Spanish Ministry of Science and Innovation, PID2020-113438RB-100/AEI/10.13039/501100011033

Overexpression of BAPT and DBTNBT genes in Taxus baccata in vitro cultures to enhance the biotechnological production of paclitaxel

Paclitaxel is one of the most effective anticancer drugs ever developed. Although the most sustainable approach to its production is provided by plant cell cultures, the yield is limited by bottleneck enzymes in the taxane biosynthetic pathway: baccatin-aminophenylpropanoyl-13-O-transferase (BAPT) and 3'-N-debenzoyltaxol N-benzoyltransferase (DBTNBT). With the aim of enhancing paclitaxel production by overcoming this bottleneck, we obtained distinct lines of Taxus baccata in vitro roots, each independently overexpressing either of the two flux-limiting genes, BAPT or DBTNBT, through a Rhizobium rhizogenes A4-mediated transformation. Due to the slow growth rate of the transgenic Taxus roots, they were dedifferentiated to obtain callus lines and establish cell suspensions. The transgenic cells were cultured in a two-stage system and stimulated for taxane production by a dual elicitation treatment with 1 μm coronatine plus 50 mm of randomly methylated-β-cyclodextrins. A high overexpression of BAPT (59.72-fold higher at 48 h) and DBTNBT (61.93-fold higher at 72 h) genes was observed in the transgenic cell cultures, as well as an improved taxane production. Compared to the wild type line (71.01 mg/L), the DBTNBT line produced more than four times higher amounts of paclitaxel (310 mg/L), while the content of this taxane was almost doubled in the BAPT line (135 mg/L). A transcriptional profiling of taxane biosynthetic genes revealed that GGPPS, TXS and DBAT genes were the most reactive to DBTNBT overexpression and the dual elicitation, their expression increasing gradually and constantly. The same genes exhibited a pattern of isolated peaks of expression in the elicited BAPT-overexpressing line.This research was funded by Agencia Estatal de Investigación. REF: PID2020-113438RBI00/AEI/10.13039/501100011033 and by the Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR) del Departament de Recerca i Universitats de la Generalitat de Catalunya 2021 SGR00693. D.H.-M. is a Postdoctoral researcher at María Zambrano at the University of Barcelona. His contract is financed by the Ministry of Universities, the European Union Next GenerationEU/PRTR.i and the Recovery, Transformation and Resilience Plan