Éditeur(s) : HAL CCSD
Résumé : Croton urucurana Baillon (Euphorbiaceae), plant species popularly known in Brazil as “sangra-d’água”, is commonly found in Mato Grosso do Sul State (midwestern Brazil), where its latex is popularly used to treat several types of cancer. Despite the ethnopharmacological significance of the species, no phytochemical or cytotoxic studies of its latex have been published. In this context, the aim of the present study was to assess the cytotoxic activity of crude extracts, fractions and isolated compound from the latex of C. urucurana and to investigate the effects of crourorb A1 on the viability, apoptosis, cell cycle and cell migration of human hepatocarcinoma cell lines (Huh-7). Also, we have evaluated the viability of Huh-7 cells treated with crourorb A1 in 3D collagen gels. The cytotoxicity was detected using the sulforhodamine B (SRB) assay against six human cancer cells lines and one nontumor NIH/3T3 cell line (murine fibroblast). Studies on the mechanism of cell death of crourorb A1 were conducted in different test systems in vitro. The bioactive ethyl acetate phase obtained from the latex of C. urucurana afforded a novel cyclic peptide, [1−9-NαC]-crourorb A1, that proved active against NCIADR/ RES (ovary, multidrug-resistance phenotype) cells with the same potency as doxorubicin (positive control) and inactive up to the highest concentration tested against nontumor NIH/3T3 cells (GI50 = > 250 μg/mL). Crourorb A1 treatment decreases the cell viability of Huh-7 at dose and time dependent manner associated with induction of apoptosis by caspase 3/7 activation and increasing expression of pro-apoptotic proteins: Bak, Bid, Bax, Puma, Bim, and Bad and clived-caspase3 protein. The JNK/MAP pathway is involved in this induced cell death. Crourorb A1 effect is also associated with G2/M phase cell cycle arrest. In parallel, we observed that crourorb A1 treatment resulted in an increase of cyclin-dependent kinase (CDK1), cyclin B1 and cyclin D1 expressions. A significant reduction of Huh-7 cells migration by crourorb A1 can be assessed and we also show that cells grown on 3D collagen gels exhibited increased resistance to crourorb A1 when compared to 2D cell culture.
Croton urucurana Baillon (Euphorbiaceae), est une plante très connue et utilisée en médecine traditionnelle au Brésil, la «sangra-d’água» retrouvée dans l'État du Mato Grosso do Sul (région Centre-Ouest du Brésil). Son latex est abondamment utilisé en médecine traditionnelle dans le traitement de plusieurs types de cancer. En dépit de son importance ethnopharmacologique, on ne trouve aucune étude sur sa composition chimique ou sur sa potentielle activité anti-cancéreuse. L’objectif de ce travail a été d'évaluer l'activité cytotoxique des extraits totaux, fractions d’extraits et de molécules purifiées, isolées du latex de C. urucurana. Nous avons étudié plus particulièrement les effets du crourorb A1 sur la viabilité, l'apoptose, le cycle cellulaire et la migration de cellules issues de carcinome hépatocellulaire humaine (Huh-7). Nous avons également évalué l’effet du crourorb A1sur la viabilité des cellules Huh-7 cultivées dans les gels de collagène en 3D. L' activité cytotoxique ont été réalisées par le test à la sulforhodamine B sur six lignées cellulaire cancéreuses et sur une lignée cellulaire non cancéreuse de fibroblastes murins, les cellules NIH/3T3. L'étude phytochimique de la fraction ‘acétate d'éthyle’ du latex de C. urucurana bioguidé a permis d’obtenir un nouveau cyclopeptide que nous avons nommé [1-9-NαC]-crourorb A1. Le crourorb présente une cytotoxicité puissante sur la lignée cellulaire cancéreuse NCI-ADR/RES (ovaire, le phénotype de multirésistance) à un niveau équivalent à celle de la doxorubicine (témoin positif). Ce composé est de plus pratiquement inactif (GI50 = >250 μg/mL) sur la lignée non cancéreuse NIH/3T3. L'évaluation de la cytotoxicité in vitro du crourorb A1 (test wst1) sur les cellules Huh-7 a démontré que la viabilité des cellules est inhibée de manière dose et temps-dépendant. Nous avons mesuré une forte activation des caspases 3 et 7 et une nette augmentation de l’expression des protéines pro-apoptotiques (Bak, Bid, Bax, Puma, Bim et Bad) ainsi que de la caspase 3 clivée par le crourorb A1. Nous avons également déterminé que la voie de signalisation JNK-MAPkinase semble nécessaire à l’induction de la mort cellulaire des cellules Huh-7 traitées au crourorb A1. Le crourob A1 induit un arrêt du cycle cellulaire en phase G2/M. En parallèle, les expressions de la cycline-dépendante kinase CDK1, des cyclines B1 et D1 sont augmentées après traitement au crourorb A1. De plus, le crourorb A1 a un effet inhibiteur significatif de la migration des cellules Huh-7, en présence ou absence de mitomycine C. Nous avons également confirmé, l’effet inhibiteur du crourob A1 sur la viabilité des cellules Huh7 cultivées en 3D dans les gels de collagène 1 et démontré que les cellules cultivées en 3D semblent plus résistantes au crourorb A1 par rapport aux cultures en monocouche 2D.
Croton urucurana Baillon (Euphorbiaceae) é uma espécie arbórea conhecida popularmente no Brasil como “sangra d´água”, comumente encontrada no estado de Mato Grosso do Sul, onde seu látex tem sido utilizado na medicina popular no tratamento de vários tipos de câncer. Apesar de sua importância etnofarmacológica, não há relatos de estudos relacionados à sua composição química ou atividades anticâncer na literatura. Nesse contexto, o objetivo do presente estudo foi avaliar a atividade citotóxica de extratos, fases, frações e da substância isolada do látex de C. ururucurana e investigar os mecanismos celulares e moleculares in vitro responsáveis pela citotoxicidade da substância isolada considerando seu potencial apoptótico e influência sobre a progressão do ciclo celular em uma linhagem de hepatocarcinoma celular humano (Huh-7), bem como, os efeitos desta substância sobre a migração e viabilidade de células Huh-7 cultivadas em geis de colágeno em 3D. O ensaio de citotoxicidade in vitro foi realizado com o corante sulforrodamina B (SRB) em seis linhagens de células neoplásicas humanas e uma linhagem de células não neoplásicas NIH/3T3 (fibloblasto murinho). Os estudos de mecanismos de ação com crourorb A1 foram conduzidos em diferentes sistemas-teste in vitro. O estudo fitoquímico biomonitorado da fase bioativa acetato de etila do látex de C. urucurana resultou no isolamento de um novo ciclopeptídeo denominado [19-NαC]-crourorb A1, que apresentou potente atividade citotóxica frente à linhagem de células neoplásicas NCI-ADR/RES (ovário com fenótipo de resistência a múltiplos fármacos), com a mesma potência que a doxorrubicina (controle positivo), e foi inativa até a maior concentração testada (GI50 = >250 μg/mL) frente à linhagem de células não neoplásicas 3T3. A exposição de células Huh-7 ao crourorb A1 resultou em uma diminuição da viabilidade celular de maneira dose e tempo-dependentes. Nós observamos um aumento da atividade de caspases 3 e 7 e um aumento da expressão de proteínas próapoptóticas (Bak, Bid, Bax, Puma, Bim e Bad) e caspase 3 clivada após tratamento com crourorb A1. Além disso, observamos que a via de sinalização celular MAP/JNK foi necessária para induzir a morte celular em células Huh-7 tratadas com crourorb A1. Os efeitos de crourorb A1 foram também associados com o acúmulo de células na fase G2/M do ciclo celular. Em paralelo, as expressões da cinase dependente da ciclina (CDK1), ciclina B1 e ciclina D1 foram aumentadas após tratamento com crourorb A1. Foi demonstrado também o envolvimento de crourorb A1 na redução da migração de células Huh-7 cultivadas na presença e ausência de mitomicina C. A avaliação da viabilidade de células Huh-7 cultivadas em geis de colágeno em 3D apontou uma maior resistência destas células ao crourorb A1 quando comparadas com a cultura de células em 2D.
https://tel.archives-ouvertes.fr/tel-01478286

NNT : 2016REN1B010
tel-01478286
https://tel.archives-ouvertes.fr/tel-01478286
https://tel.archives-ouvertes.fr/tel-01478286/document
https://tel.archives-ouvertes.fr/tel-01478286/file/DE-MATOS-CANDIDO-BACANI-Priscila.pdf

Éditeur(s) : HAL CCSD Wiley
Résumé : International audience
Mechanical forces influence the growth and shape of virtually all tissues and organs. Recent studies show that increased cell contractibility, growth and differentiation might be normalized by modulating cell tensions. Particularly, the role of these tensions applied by the extracellular matrix during liver fibrosis could influence the hepatocarcinogenesis process. The objective of this study is to determine if 3D stiffness could influence growth and phenotype of normal and transformed hepatocytes and to integrate extracellular matrix (ECM) stiffness to tensional homeostasis. We have developed an appropriate 3D culture model: hepatic cells within three-dimensional collagen matrices with varying rigidity. Our results demonstrate that the rigidity influenced the cell phenotype and induced spheroid clusters development whereas in soft matrices, Huh7 transformed cells were less proliferative, well-spread and flattened. We confirmed that ERK1 played a predominant role over ERK2 in cisplatin-induced death, whereas ERK2 mainly controlled proliferation. As compared to 2D culture, 3D cultures are associated with epithelial markers expression. Interestingly, proliferation of normal hepatocytes was also induced in rigid gels. Furthermore, biotransformation activities are increased in 3D gels, where CYP1A2 enzyme can be highly induced/activated in primary culture of human hepatocytes embedded in the matrix. In conclusion, we demonstrated that increasing 3D rigidity could promote proliferation and spheroid developments of liver cells demonstrating that 3D collagen gels are an attractive tool for studying rigidity-dependent homeostasis of the liver cells embedded in the matrix and should be privileged for both chronic toxicological and pharmacological drug screening.
ISSN: 0730-2312

hal-01196600
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01196600
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01196600/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01196600/file/Increasing%203D%20Matrix%20Rigidity_accepted.pdf
DOI : 10.1002/jcb.25356

Éditeur(s) : HAL CCSD Elsevier
Résumé : Thanks to Dr M. Blanchard-Desce (UMR CNRS 5255, University of Bordeaux, France), who kindly provided us with fluorescent dye RS19 and the PIXEL, MRIc, flow cytometry and H2P2 platforms (SFR Biosit, UMS CNRS 3480/US INSERM 018, Rennes).
International audience
[1-9-NαC]-crourorb A1 is a cyclic peptide isolated from Croton urucurana Baillon latex, found in midwestern Brazil, that has been shown to exert cytotoxic effects against a panel of cancer cell lines. However, the underlying mechanisms responsible for the crourorb A1-induced cytotoxicity in cancer cells remain unknown. In this study, the effects of crourorb A1 on the viability, apoptosis, cell cycle and migration of Huh-7 (human hepatocarcinoma) cells were investigated. We evaluated the viability of Huh-7 cells treated with crourorb A1 in 2D and 3D collagen cultures and found that cells in 3D culture exhibited increased resistance to crourorb A1 compared to cells in 2D culture (IC50: 62μg/ml versus 35.75μg/ml). Crourorb A1 treatment decreases the viability of Huh-7 cells in a dose- and time-dependent manner and is associated with the induction of apoptosis, in the absence of necrotic cells, through the activation of caspase-3/7 and increased expression of the pro-apoptotic proteins Bak, Bid, Bax, Puma, Bim, and Bad. The effects of crourorb A1 are also associated with G2/M phase cell cycle arrest and increases in cyclin-dependent kinase (CDK1) and cyclin B1 expression. A significant reduction in Huh-7 cell migration induced by crourorb A1 was also observed in the presence of mitomycin C. Finally, we showed that the JNK/MAP pathway, but not ERK signaling, is involved in crourorb A1-induced hepatocarcinoma cell mortality.
ISSN: 0378-4274

hal-01526429
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01526429
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01526429/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01526429/file/de%20Matos%20C%C3%A2ndido-Bacani%20et%20al.%20-%20%5B1%E2%80%939-N%CE%B1C%5D-crourorb%20A1%20isolated%20from%20Croton%20urucura.pdf
DOI : 10.1016/j.toxlet.2017.03.020

Éditeur(s) : HAL CCSD Public Library of Science
Résumé : International audience
Among persisting questions on bone calcification, a major one is the link between protein expression and mineral deposition. A cell culture system is here proposed opening new integrative studies on biomineralization, improving our knowledge on the role played by non-collagenous proteins in bone. This experimental in vitro model consisted in human primary osteoblasts cultured for 60 days at the surface of a 3D collagen scaffold mimicking an osteoid matrix. Various techniques were used to analyze the results at the cellular and molecular level (adhesion and viability tests, histology and electron microscopy, RT-and qPCR) and to characterize the mineral phase (histological staining, EDX, ATG, SAED and RMN). On long term cultures human bone cells seeded on the osteoid-like matrix displayed a clear osteoblast phenotype as revealed by the osteoblast-like morphology, expression of specific protein such as alkaline phosphatase and expression of eight genes classically considered as osteoblast markers, including BGLAP, COL1A1, and BMP2. Von Kossa and alizarine red allowed us to identify divalent calcium ions at the surface of the matrix, EDX revealed the correct Ca/P ratio, and SAED showed the apatite crystal diffraction pattern. In addition RMN led to the conclusion that contaminant phases were absent and that the hydration state of the mineral was similar to fresh bone. A temporal correlation was established between quantified gene expression of DMP1 and IBSP, and the presence of hydroxyapatite, confirming the contribution of these proteins to the mineralization process. In parallel a difference was observed in the expression pattern of SPP1 and BGLAP, which questioned their attributed role in the literature. The present model opens new experimental possibilities to study spatio-temporal relations between bone cells, dense collagen scaffolds, NCPs and hydroxyapatite mineral deposition. It also emphasizes the importance of high collagen density environment in bone cell physiology.
ISSN: 1932-6203

hal-01142641
http://hal.upmc.fr/hal-01142641
http://hal.upmc.fr/hal-01142641/document
http://hal.upmc.fr/hal-01142641/file/Silvent_2013_Collagen.pdf
DOI : 10.1371/journal.pone.0057344

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
From 15 to 17 June 2011, a dedicated workshop was held on the subject of in vitro models for mammalian spermatogenesis and their applications in toxicological hazard and risk assessment. The workshop was sponsored by the Dutch ASAT initiative (Assuring Safety without Animal Testing), which aims at promoting innovative approaches toward toxicological hazard and risk assessment on the basis of human and in vitro data, and replacement of animal studies. Participants addressed the state of the art regarding human and animal evidence for compound mediated testicular toxicity, reviewed existing alternative assay models, and brainstormed about future approaches, specifically considering tissue engineering. The workshop recognized the specific complexity of testicular function exemplified by dedicated cell types with distinct functionalities, as well as different cell compartments in terms of microenvironment and extracellular matrix components. This complexity hampers quick results in the realm of alternative models. Nevertheless, progress has been achieved in recent years, and innovative approaches in tissue engineering may open new avenues for mimicking testicular function in vitro. Although feasible, significant investment is deemed essential to be able to bring new ideas into practice in the laboratory. For the advancement of in vitro testicular toxicity testing, one of the most sensitive end points in regulatory reproductive toxicity testing, such an investment is highly desirable.
ISSN: 0890-6238

hal-00876979
https://hal.archives-ouvertes.fr/hal-00876979
DOI : 10.1016/j.reprotox.2013.04.004
PUBMED : 23612449