Hepatoprotective, Antioxidant, and Anticancer Effects of the Tragopogon porrifolius

Tragopogon porrifolius (Asteraceae), commonly referred to as white salsify, is an edible herb used in Lebanese folk medicine to treat cancer and liver dysfunction. In this study, we investigated the antioxidant activity of Tragopogon porrifolius methanolic extract, both in vitro and in vivo, in addition to its hepatoprotective and anticancer activities. Total phenolic and flavonoid contents were measured and found to be  mg GAE/g and  mg QE/g dry weight, respectively. In vitro antioxidant assays revealed an FRAP value of  µmol Fe2+/g of extract and DPPH IC50 value 15.2 µg/mL. In rats subjected to CCl4-induced hepatotoxicity, significant increase in CAT, SOD, and GST levels was detected. The highest dose of the extract (250 mg/kg) recorded a fold increase of 1.68 for SOD, 2.49 for GST, and 3.2 for CAT. The extract also showed substantial decrease in AST (57%), ALT (56%), and LDH (65%) levels. Additionally, the extract caused a dose-dependent decrease in cell viability and proliferation. In conclusion, the methanolic extract of T. porrifolius displayed a relatively high antioxidant activity both in vitro and in vivo as well as hepatoprotective potential against liver toxicity in rats and anticancer effect on MDA-MB-231 and Caco-2 cells.PublishedN/

The functional interplay between eIF2alpha phosphorylation and mTOR signaling pathways: implications in Tuberous Sclerosis Complex disorder

The mammalian target of rapamycin (mTOR) nucleates two complexes, namely mTOR complex 1 (mTORC1) and mTORC2, which are implicated in cell growth, survival, metabolism and cancer. The phosphorylation of the alpha subunit of the eukaryotic initiation factor eIF2 at serine 51 (eIF2αP) is a key regulator of mRNA translation and an important mechanism of adaptation of cells to various forms of environmental stress frequently associated with cancer formation. eIF2αP can act either as a promoter of cell survival or as an inducer of cell death in response to different forms of stress. Increased eIF2αP is mediated by a family of four kinases consisting of PKR, PERK, GCN2 and HRI, each of which becomes activated by distinct stimuli. In this study, we show that disruption of mTORC2, but not of mTORC1, induces eIF2αP through the activation of PERK. mTORC2 deficiency increases PERK activity owing to the ineffecive activation of AKT, which negatively controls PERK by phosphorylation at threonine 799. Moreover, pharmacological inhibition of mTOR with either rapamycin or the new generation of catalytic inhibitors also increases PERK activity and eIF2αP. Interestingly, rapamycin treatment induces eIF2αS51P through a mechanism that is independent of mTORC1 inhibition.The physiological relevance of our findings was substantiated in cells deficient in tuberous sclerosis complex (TSC), which have impaired mTORC2/AKT function, but increased mTORC1 activity. Our research shows that TSC-deficient cells exposed to ER stress exhibit increased levels of the PERK-eIF2αP arm, which functions as a compensatory mechanism to substitute for the loss of AKT and facilitate cell survival. TSC-deficient cells subjected to oxidative stress on the other hand, downregulate PERK-eIF2αP but activate the PKR-eIF2αP arm instead in an mTORC1-S6K1-mediated mechanism to promote cell death. Furthermore, we show that TSC-null cells deficient in eIF2αP have a greater tolerance to oxidative stress, leading to an increase in their tumorigenic potential and therefore allowing an earlier tumor incidence. Our study reveals that eIF2αP acts downstream of either mTORC2-AKT or mTORC1-S6K1 to promote either the survival or death of TSC-mutant cells in response to different stress-inducing drugs.La cible de la rapamycine chez les mammifères (mTOR) forme deux complexes, à savoir les complexes mTOR 1 (mTORC1) et 2 (mTORC2), lesquels sont impliqués dans la prolifération cellulaire, la survie, le métabolisme et le cancer. La phosphorylation de la sous-unité alpha du facteur d’initiation eucaryote eIF2 au niveau de la sérine 51 (eIF2αP) est un régulateur essentiel de la traduction des ARNm et un mécanisme important de l’adaptation des cellules face aux diverses formes de stress environnementaux fréquemment associés à la formation du cancer. eIF2αP peut agir comme promoteur de la survie cellulaire ou bien comme inducteur de la mort cellulaire en réponse aux différentes formes de stress. L’augmentation d’eIF2αP est médiée par une famille de quatre kinases constituée par PKR, PERK, GCN2 et HRI, chacune d’entre elles étant activée par des stimuli distincts. Dans cette étude, nous montrons que la perte de mTORC2, et non pas de mTORC1, induit eIF2αP via l’activation de PERK. L’absence de mTORC2 augmente l’activité de PERK en raison de l’activation insuffisante d’AKT, lequel contrôle négativement PERK par phosphorylation à la thréonine 799. De plus, l’inhibition pharmacologique de mTOR par la rapamycine ou par la nouvelle génération d’inhibiteurs catalytiques augmente aussi l’activité de PERK et eIF2αP. Fait intéressant, le traitement à la rapamycine induit eIF2αS51P par le biais d’un mécanisme indépendant de l’inhibition de mTORC1.La pertinence physiologique de nos résultats a été mise en évidence dans les cellules déficientes en sclérose tubéreuse complexe (TSC), lesquelles ont une fonction altérée de mTORC2/AKT mais qui présentent une augmentation de l’activité de mTORC1. Nos recherches montrent que les cellules déficientes en TSC exposées à un stress du réticulum endoplasmique (ER) augmentent les niveaux de la voie PERK-eIF2αP qui sert alors de mécanisme compensatoire pour remplacer la perte d’AKT et faciliter la survie cellulaire. D’autre part, les cellules déficientes en TSC assujetties à un stress oxydatif diminuent la voie PERK-eIF2αP mais active par contre celle de PKR-eIF2αP par un mécanisme médié par mTORC1-S6K1 pour promouvoir la mort cellulaire. Par ailleurs, nous montrons que les cellules TSC-nulles déficientes en eIF2αP ont une plus grande tolérance au stress oxydatif, ce qui conduit à augmenter leur potentiel tumorigène et donc permettre une incidence plus précoce des tumeurs. Notre étude révèle qu’eIF2αP agit en aval de mTORC2-AKT ou de mTORC1-S6K1 pour promouvoir la survie ou la mort des cellules déficientes en TSC en réponse aux différents traitements induisant le stress

Species-, organ- and cell-type-dependent expression of SPARCL1 in human and mouse tissues

SPARCL1 is a matricellular protein with anti-adhesive, anti-proliferative and anti-tumorigenic functions and is frequently downregulated in tumors such as colorectal carcinoma or non-small cell lung cancer. Studies have identified SPARCL1 as an angiocrine tumor suppressor secreted by tumor vessel endothelial cells, thereby exerting inhibitory activity on angiogenesis and tumor growth, in colorectal carcinoma. It is unknown whether SPARCL1 may exert these homeostatic functions in all organs and in other species. Therefore, SPARCL1 expression was comparatively analysed between humans and mice in a systematic manner. Murine Sparcl1 (mSparcl1) is most strongly expressed in the lung; expressed at an intermediate level in most organs, including the large intestine; and absent in the liver. In human tissues, SPARCL1 (hSPARCL1) was detected in all organs, with the strongest expression in the stomach, large intestine and lung, mostly consistent with the murine expression pattern. A striking difference between human and murine tissues was the absence of mSparcl1 expression in murine livers, while human livers showed moderate expression. Furthermore, mSparcl1 was predominantly associated with mural cells, whereas hSPARCL1 was detected in both mural and endothelial cells. Human SPARCL1 expression was downregulated in different carcinomas, including lung and colon cancers. In conclusion, this study revealed species-, organ- and cell-type-dependent expression of SPARCL1, suggesting that its function may not be similar between humans and mice

IFN-γ-response mediator GBP-1 represses human cell proliferation by inhibiting the Hippo signaling transcription factor TEAD

Interferon-gamma (IFN-γ) is a pleiotropic cytokine that exerts important functions in inflammation, infectious diseases, and cancer. The large GTPase human guanylate-binding protein 1 (GBP-1) is among the most strongly IFN-γ-induced cellular proteins. Previously, it has been shown that GBP-1 mediates manifold cellular responses to IFN-γ including the inhibition of proliferation, spreading, migration, and invasion and through this exerts anti-tumorigenic activity. However, the mechanisms of GBP-1 anti-tumorigenic activities remain poorly understood. Here, we elucidated the molecular mechanism of the human GBP-1-mediated suppression of proliferation by demonstrating for the first time a cross-talk between the anti-tumorigenic IFN-γ and Hippo pathways. The α9-helix of GBP-1 was found to be sufficient to inhibit proliferation. Protein-binding and molecular modeling studies revealed that the α9-helix binds to the DNA-binding domain of the Hippo signaling transcription factor TEA domain protein (TEAD) mediated by the 376VDHLFQK382 sequence at the N-terminus of the GBP-1-α9-helix. Mutation of this sequence resulted in abrogation of both TEAD interaction and suppression of proliferation. Further on, the interaction caused inhibition of TEAD transcriptional activity associated with the down-regulation of TEAD-target genes. In agreement with these results, IFN-γ treatment of the cells also impaired TEAD activity, and this effect was abrogated by siRNA-mediated inhibition of GBP-1 expression. Altogether, this demonstrated that the α9-helix is the proliferation inhibitory domain of GBP-1, which acts independent of the GTPase activity through the inhibition of the Hippo transcription factor TEAD in mediating the anti-proliferative cell response to IFN-γ

mTORC1 and CK2 coordinate ternary and eIF4F complex assembly

International audienceTernary complex (TC) and eIF4F complex assembly are the two major rate-limiting steps in translation initiation regulated by eIF2α phosphorylation and the mTOR/4E-BP pathway, respectively. How TC and eIF4F assembly are coordinated, however, remains largely unknown. We show that mTOR suppresses translation of mRNAs activated under short-term stress wherein TC recycling is attenuated by eIF2α phosphorylation. During acute nutrient or growth factor stimulation, mTORC1 induces eIF2β phosphorylation and recruitment of NCK1 to eIF2, decreases eIF2α phosphorylation and bolsters TC recycling. Accordingly, eIF2β mediates the effect of mTORC1 on protein synthesis and proliferation. In addition, we demonstrate a formerly undocumented role for CK2 in regulation of translation initiation, whereby CK2 stimulates phosphorylation of eIF2β and simultaneously bolsters eIF4F complex assembly via the mTORC1/4E-BP pathway. These findings imply a previously unrecognized mode of translation regulation, whereby mTORC1 and CK2 coordinate TC and eIF4F complex assembly to stimulate cell proliferation