Pregnane × Receptor (PXR) expression in colorectal cancer cells restricts irinotecan chemosensitivity through enhanced SN-38 glucuronidation

<p>Abstract</p> <p>Background</p> <p>Clinical efficacy of chemotherapy in colorectal cancer is subjected to broad inter-individual variations leading to the inability to predict outcome and toxicity. The topoisomerase I inhibitor irinotecan (CPT-11) is worldwide approved for the treatment of metastatic colorectal cancer and undergoes extensive peripheral and tumoral metabolism. PXR is a xenoreceptor activated by many drugs and environmental compounds regulating the expression of drug metabolism and transport genes in detoxification organs such as liver and gastrointestinal tract. Considering the metabolic pathway of irinotecan and the tissue distribution of Pregnane × Receptor (PXR), we hypothesized that PXR could play a key role in colon cancer cell response to irinotecan.</p> <p>Results</p> <p>PXR mRNA expression was quantified by RT-quantitative PCR in a panel of 14 colon tumor samples and their matched normal tissues. PXR expression was modulated in human colorectal cancer cells LS174T, SW480 and SW620 by transfection and siRNA strategies. Cellular response to irinotecan and its active metabolic SN38 was assessed by cell viability assays, HPLC metabolic profiles and mRNA quantification of PXR target genes. We showed that PXR was strongly expressed in colon tumor samples and displayed a great variability of expression. Expression of hPXR in human colorectal cancer cells led to a marked chemoresistance to the active metabolite SN38 correlated with PXR expression level. Metabolic profiles of SN38 showed a strong enhancement of SN38 glucuronidation to the inactive SN38G metabolite in PXR-expressing cells, correlated with an increase of UDPglucuronosyl transferases UGT1A1, UGT1A9 and UGT1A10 mRNAs. Inhibition of PXR expression by lentivirus-mediated shRNA, led to SN38 chemoresistance reversion concomitantly to a decrease of UGT1A1 expression and SN38 glucuronidation. Similarly, PXR mRNA expression levels correlated to UGT1A subfamily expression in human colon tumor biopsies.</p> <p>Conclusion</p> <p>Our results demonstrate that tumoral metabolism of SN38 is affected by PXR and point to potential therapeutic significance of PXR quantification in the prediction of irinotecan response. Furthermore, our observations are pharmacologically relevant since many patients suffering from cancer diseases are often exposed to co-medications, food additives or herbal supplements able to activate PXR. A substantial part of the variability observed among patients might be caused by such interactions</p

Active site specificity profiling of the matrix metalloproteinase family: Proteomic identification of 4300 cleavage sites by nine MMPs explored with structural and synthetic peptide cleavage analyses

Secreted and membrane tethered matrix metalloproteinases (MMPs) are key homeostatic proteases regulating the extracellular signaling and structural matrix environment of cells and tissues. For drug targeting of proteases, selectivity for individual molecules is highly desired and can be met by high yield active site specificity profiling. Using the high throughput Proteomic Identification of protease Cleavage Sites (PICS) method to simultaneously profile both the prime and non-prime sides of the cleavage sites of nine human MMPs, we identified more than 4300 cleavages from P6 to P6′ in biologically diverse human peptide libraries. MMP specificity and kinetic efficiency were mainly guided by aliphatic and aromatic residues in P1′ (with a ~ 32–93% preference for leucine depending on the MMP), and basic and small residues in P2′ and P3′, respectively. A wide differential preference for the hallmark P3 proline was found between MMPs ranging from 15 to 46%, yet when combined in the same peptide with the universally preferred P1′ leucine, an unexpected negative cooperativity emerged. This was not observed in previous studies, probably due to the paucity of approaches that profile both the prime and non-prime sides together, and the masking of subsite cooperativity effects by global heat maps and iceLogos. These caveats make it critical to check for these biologically highly important effects by fixing all 20 amino acids one-by-one in the respective subsites and thorough assessing of the inferred specificity logo changes. Indeed an analysis of bona fide MEROPS physiological substrate cleavage data revealed that of the 37 natural substrates with either a P3-Pro or a P1′-Leu only 5 shared both features, confirming the PICS data. Upon probing with several new quenched-fluorescent peptides, rationally designed on our specificity data, the negative cooperativity was explained by reduced non-prime side flexibility constraining accommodation of the rigidifying P3 proline with leucine locked in S1′. Similar negative cooperativity between P3 proline and the novel preference for asparagine in P1 cements our conclusion that non-prime side flexibility greatly impacts MMP binding affinity and cleavage efficiency. Thus, unexpected sequence cooperativity consequences were revealed by PICS that uniquely encompasses both the non-prime and prime sides flanking the proteomic-pinpointed scissile bond

Etude du phénomène d'interférence dans l'accord sujet-verbe chez l'adulte

Les productions langagières des locuteurs sont le lieu d’erreurs d’accord entre le sujet et le verbe dans des phrases contenant un objet interférent, ayant un nombre distinct de celui du sujet (par exemple, *Voilà les filles que le voisin ONT rencontrées). Diverses théories syntaxiques proposent l’idée que lorsque l’objet est en début de phrase, il s’est déplacé au sein de sa structure syntaxique en passant par des positions intermédiaires, non prononcées à l’oral. A quel niveau s’effectue cette interférence dans l’accord ? L’hypothèse des traces intermédiaires (Franck, Soare, Frauenfelder, & Rizzi, 2010) suppose que cette interférence a lieu au niveau des traces intermédiaires de l’objet spécifiquement, tandis qu’une hypothèse alternative d’activation générale, suggère que cette interférence a lieu tout au long du déplacement de l’objet jusqu’à sa position finale. Cette recherche vise à départager les deux hypothèses. Une première expérience étudiait l’interférence au sein de phrases du type « Voilà les médecins que, quand l’enfant A guéri, le père EST allé remercier. », en contrastant deux types de structure : les participants, 24 adultes francophones, accordaient soit le verbe de la proposition principale (EST / SONT), soit celui de l’ajout (A / ONT), avec la procédure de compréhension de présentation visuelle sérielle rapide. Les phrases utilisées contenant des virgules, une seconde expérience, dont le matériel est mélangé à la première, visait à vérifier que les virgules n’isolent pas le traitement de la partie qu’elles contiennent. Les structures manipulées étaient des propositions relatives objet soit restrictives (par exemple, Les assistants que le doyen A / ONT convoqués sont agacé.), soit appositives (par exemple, Mes assistants, que le doyen A / ONT convoqués, sont agacés.). Un effet d’interférence avec l’objet a été trouvé dans l’expérience « Relative Restrictive vs. Appositive », similaire dans les deux conditions, et répliquant diverses recherches précédentes avec un nouveau matériel et une nouvelle procédure. Toutefois, contrairement à nos attentes, aucun effet n’est apparu dans l’expérience « Ajout vs. Principale ». Les résultats ne permettent donc pas de départager les deux hypothèses. Les différents biais possibles pouvant expliquer cette absence d’effet sont discutés, puis une réflexion en perspective d’une recherche future est proposée à la fin de ce travail

Rôle des lipoxines dans la régulation de l'inflammation dans l'asthme

MONTPELLIER-BU Médecine UPM (341722108) / SudocPARIS-BIUP (751062107) / SudocMONTPELLIER-BU Médecine (341722104) / SudocSudocFranceF

Real-time imaging of myeloid cells dynamics in ApcMin/+ intestinal tumors by spinning disk confocal microscopy.

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. Apc(Min/+) mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment

Lipid Mediators as Agonists for the Resolution of Acute Lung Inflammation and Injury

Resolution of acute lung inflammation and injury is an active process; it is not merely the absence of proinflammatory signals. Restoration of homeostasis is coordinated by specific mediators and cellular events. In response to injury and inflammatory stimuli, infiltrating leukocytes and tissue-resident cells interact to generate lipoxins (LXs), which are bioactive eicosanoids derived from arachidonic acid. In contrast to proinflammatory leukotrienes and prostaglandins, LXs display potent antiinflammatory actions. LXA4 interacts with a G protein–coupled receptor, termed ALX, that transduces counter-regulatory signals in part via intracellular polyisoprenyl phosphate remodeling. Presqualene diphosphate (PSDP) is a polyisoprenyl phosphate in human neutrophils that is rapidly converted to presqualene monophosphate (PSMP) upon cell activation. PSDP, but not PSMP, directly inhibits phospholipase D, phosphoinositol-3 kinase, and superoxide anion generation. LXs block PSDP turnover in neutrophil membranes to prevent proinflammatory responses. Hence, LX and polyisoprenyl phosphate signaling provide a counter-regulatory circuit to promote resolution of acute lung inflammation. LXA4 and PSDP mimetics have been prepared with potent protective actions in murine models of asthma and acute lung injury