Pauline Poinot

Enhancing tumor response to targeted chemotherapy through up-regulation of folate receptor α expression induced by dexamethasone and valproic acid

Abstract Several folate‐drug conjugates are currently undergoing clinical trials for application in oncology. However, the efficacy of folate‐targeted therapy strongly depends on the folate receptor (FR) abundance at the surface of cancer cells. Recently, it has been postulated that up‐regulation of FR&agr; by means of chemo‐sensitizing agents could enhance the anticancer activity of FR‐drug conjugates. In this study, we demonstrate in vitro that a combination of dexamethasone (Dexa) and valproic acid (VPA) increases FR&agr; expression selectively at the surface of FR‐overexpressing cancer cells. The same stimulation was observed in vivo in KB‐tumor xenografts when mice are treated with this combined treatment. This effect is reversible since treatment interruption induces the return of FR expression at basal level. When incubated with Dexa and VPA, the &bgr;‐galactosidase‐responsive folate‐monomethyl auristatin E (MMAE) conjugate, called MGAF, exhibits higher cytotoxic activity on several FR‐positive human cancer cell lines, compared to its administration as a single agent. This improved toxicity results from the enhanced concentration of MMAE released within cancer cells after internalization and subsequent enzymatic activation of MGAF. Higher deposition of MMAE is also observed in vivo after up‐regulation of FR expression level in tumor xenografts, induced by the prior administration of the Dexa/VPA combination. In this model, MGAF/Dexa/VPA combined therapy results in an 81% inhibition of tumor growth compared to the control group, while MGAF used in monotherapy is inefficient. Since Dexa and VPA are currently used in humans, this finding could be of great interest for further development of folate‐drug conjugates, in particular for those that are presently under clinical investigation. Graphical abstract Figure. No Caption available.

Study of a novel agent for TCA precipitated proteins washing - comprehensive insights into the role of ethanol/HCl on molten globule state by multi-spectroscopic analyses

Sample preparation for mass spectrometry-based proteomics is a key step for ensuring reliable data. In gel-free experimental workflows, protein purification often starts with a precipitation stage using trichloroacetic acid (TCA). In presence of TCA, proteins precipitate in a stable molten globule state making the pellet difficult to solubilize in aqueous buffer for proteolytic digestion and MS analysis. In this context, the objective of this work was to study the suitability of a novel agent, ethanol/HCl, for the washing of TCA-precipitated proteins. This method optimized the recovery of proteins in aqueous buffer (50 to 96%) while current organic solvents led to losses of material. Following a mechanistic study, the effect of ethanol/HCl on the conformation of TCA-precipitated proteins was investigated. It was shown that the reagent triggered the unfolding of TCA-stabilized molten globule into a reversible intermediate, characterized by a specific Raman signature, which favored protein subsequent resolubilization. Finally, the efficiency of ethanol/HCl for the washing of TCA-precipitated proteins extracted from a biofilm, a soil or a mouse liver was demonstrated (data available via ProteomeXchange with identifier PXD008110). Being versatile and simple, it could be of great interest to include an ethanol/HCl wash-step to produce high-quality protein extracts. SIGNIFICANCE In mass spectrometry-based proteomics workflows, proteins precipitation and/or washing usually involves the use of acetone. In fact, this solvent is effective for removing both biological interferences (e.g. lipids) and chemicals employed in protein extraction/purification protocols (e.g. TCA, SDS). However, the use of acetone can lead to significant protein losses. Moreover, when proteins are precipitated with TCA, the acetone-treated precipitate remains hard to disperse, leading to poor resolubilization of proteins in aqueous buffers. Here, we investigated the use of ethanol/HCl for washing TCA-precipitated proteins, with the aim to produce high-quality protein extracts which can be directly analyzed by LC-MS. An opening study on standard solutions showed that ethanol/HCl led to reduced losses of proteins compared to usual solvents (i.e. acetone and ethanol). This reagent also enabled a better solubilization of proteins in aqueous buffer that is necessary for their direct trypsin digestion and LC-HRMS analysis. A mechanistic study, performed through several spectroscopic analyses (LC-HRMS, Raman, spectrofluorometry), showed that treatment with ethanol/HCl induced conformational changes of TCA-precipitated proteins. Finally, we compared the efficiency of ethanol/HCl to published protocols for the washing of protein extracts from three different complex samples (i.e. soil, biofilm, and mouse liver). Our results demonstrated that ethanol/HCl is a valuable alternative to previous protein washing methods and, therefore could become a useful tool in mass spectrometry-based proteomics workflows for various applications (e.g. clinical research, chemical biology, environmental metaproteomics…).

Development of liquid chromatography high resolution mass spectrometry strategies for the screening of complex organic matter: Application to astrophysical simulated materials

The present work aims at developing two LC-HRMS setups for the screening of organic matter in astrophysical samples. Their analytical development has been demonstrated on a 100-µg residue coming from the photo-thermo chemical processing of a cometary ice analog produced in laboratory. The first 1D-LC-HRMS setup combines a serially coupled columns configuration with HRMS detection. It has allowed to discriminate among different chemical families (amino acids, sugars, nucleobases and oligopeptides) in only one chromatographic run without neither a priori acid hydrolysis nor chemical derivatisation. The second setup is a dual-LC configuration which connects a series of trapping columns with analytical reverse-phase columns. By coupling on-line these two distinct LC units with a HRMS detection, high mass compounds (350 <m/z < 600) have been efficiently preconcentrated, separated and detected. Our strategies demonstrate a real interest for the analysis of astrophysical samples coming in minute quantities and allowing only few analytical runs. Besides its relevance for astrobiological studies, this work points out the suitability of these two novel LC-HRMS strategies for untargeted analysis of complex environmental samples.

A Β-glucuronidase-responsive albumin-binding prodrug programmed for the double release of monomethylauristatin E

We report on the synthesis, in vitro and in vivo biological evaluations of a dimeric β-glucuronidase-responsive albumin-binding prodrug designed for the double release of MMAE upon a single enzymatic activation step. This prodrug produced a significant antitumour activity in mice bearing subcutaneous LS174T colorectal adenocarcinoma xenografts without inducing side effects.

TCA precipitation and ethanol/HCl single-step purification evaluation: One-dimensional gel electrophoresis, bradford assays, spectrofluorometry and Raman spectroscopy data on HSA, Rnase, lysozyme - Mascots and Skyline data

The data presented here are related to the research paper entitled “Study of a Novel Agent for TCA Precipitated Proteins Washing - Comprehensive Insights into the Role of Ethanol/HCl on Molten Globule State by Multi-Spectroscopic Analyses” (Eddhif et al., submitted for publication) [1]. The suitability of ethanol/HCl for the washing of TCA-precipitated proteins was first investigated on standard solution of HSA, cellulase, ribonuclease and lysozyme. Recoveries were assessed by one-dimensional gel electrophoresis, Bradford assays and UPLC-HRMS. The mechanistic that triggers protein conformational changes at each purification stage was then investigated by Raman spectroscopy and spectrofluorometry. Finally, the efficiency of the method was evaluated on three different complex samples (mouse liver, river biofilm, loamy soil surface). Proteins profiling was assessed by gel electrophoresis and by UPLC-HRMS.