Claude Villard

Human tumor nanoparticles induce apoptosis of pancreatic cancer cells

Exosomes are vesicles secreted by most hematopoietic cells on fusion of multivesicular endosomes with the plasma membrane. Many studies have reported that exosomes may also be released by tumor cells. Exosomes are believed to play an antitumor role through immune cells. We asked whether tumor exosomes have biological activities on tumor cells. We report that human pancreatic tumor nanoparticles, exosome‐like as characterized by proteomic analyses and rich in lipid rafts, decreased tumor cell proliferation. Nanoparticles increased Bax and decreased Bcl‐2 expressions. Caspase‐3 and −9 but not caspase‐8 inhibitors impaired apoptosis, which implicates the mitochondria apoptotic pathway. The ceramide‐sphingomyelin apoptotic pathway was inoperative. Moreover, nanoparticles induced phosphatase and tensin homolog (PTEN) and glycogen synthase kinase (GSK) −3β activation and decreased pyruvate dehydrogenase activity. In nanoparticle‐treated cells, PTEN formed complexes with actin, β‐catenin, and GSK‐3β. Thus, β‐catenin may no longer be available to activate the survival pathway. Nanoparticles triggered the down‐regulation of cyclin D1 and poly(ADP‐ribose) polymerase. Hence, nanoparticles counteracted the constitutively activated phosphatidylinositol 3‐kinase/Akt survival pathway to drive tumor cells toward apoptosis. Our study provides the first evidence of an apoptotic function of tumor‐derived nanoparticles on tumor cells. We propose a new role for nanoparticles, i.e., as signal carriers for interaction between cells, which may have implications in physiopathological situations.—Ristorcelli, E., Beraud, E., Verrando, P., Villard, C., Lafitte, D., Sbarra, V., Lombardo, D., Verine, A. Human tumor nanoparticles induce apoptosis of pancreatic cancer cells. FASEB J. 22, 3358–3369 (2008)

An Early Postnatal Oxytocin Treatment Prevents Social and Learning Deficits in Adult Mice Deficient for Magel2, a Gene Involved in Prader-Willi Syndrome and Autism

BACKGROUND Mutations of MAGEL2 have been reported in patients presenting with autism, and loss of MAGEL2 is also associated with Prader-Willi syndrome, a neurodevelopmental genetic disorder. This study aimed to determine the behavioral phenotype of Magel2-deficient adult mice, to characterize the central oxytocin (OT) system of these mutant mice, and to test the curative effect of a peripheral OT treatment just after birth. METHODS We assessed the social and cognitive behavior of Magel2-deficient mice, analyzed the OT system of mutant mice treated or not by a postnatal administration of OT, and determined the effect of this treatment on the brain. RESULTS Magel2 inactivation induces a deficit in social recognition and social interaction and a reduced learning ability in adult male mice. In these mice, we reveal anatomical and functional modifications of the OT system and show that these defects change from birth to adulthood. Daily administration of OT in the first postnatal week was sufficient to prevent deficits in social behavior and learning abilities in adult mutant male mice. We show that this OT treatment partly restores a normal OT system. Thus, we report that an alteration of the OT system around birth has long-term consequences on behavior and on cognition. Importantly, an acute OT treatment of Magel2-deficient pups has a curative effect. CONCLUSIONS Our study reveals that OT plays a crucial role in setting social behaviors during a period just after birth. An early OT treatment in this critical period could be a novel therapeutic approach for the treatment of neurodevelopmental disorders such as Prader-Willi syndrome and autism.

Advances in top-down proteomics for disease biomarker discovery

Top-down mass spectrometry strategies allow identification and characterization of proteins and protein networks by direct fragmentation. These analytical processes involve a panel of fragmentation mechanisms, some of which preserve protein post-translational modifications. Thus top-down is of special interest in clinical biochemistry to probe modified proteins as potential disease biomarkers. This review describes separating methods, mass spectrometry instrumentation, bioinformatics, and theoretical aspects of fragmentation mechanisms used for top-down analysis. The biological interest of this strategy is extensively reported regarding the characterization of post-translational modifications in biochemical pathways and the discovery of biomarkers. One has to bear in mind that quantitative aspects that are beyond the focus of this review are also of critical important for biomarker discovery. The constant evolution of technologies makes top-down strategies crucial players in clinical and basic proteomics.

Microtubule targeting agents: from biophysics to proteomics

This review explores various aspects of the interaction between microtubule targeting agents and tubulin, including binding site, affinity, and drug resistance. Starting with the basics of tubulin polymerization and microtubule targeting agent binding, we then highlight how the three-dimensional structures of drug–tubulin complexes obtained on stabilized tubulin are seeded by precise biological and biophysical data. New avenues opened by thermodynamics analysis, high throughput screening, and proteomics for the molecular pharmacology of these drugs are presented. The amount of data generated by biophysical, proteomic and cellular techniques shed more light onto the microtubule–tubulin equilibrium and tubulin–drug interaction. Combining these approaches provides new insight into the mechanism of action of known microtubule interacting agents and rapid in-depth characterization of next generation molecules targeting the interaction between microtubules and associated modulators of their dynamics. This will facilitate the design of improved and/or alternative chemotherapies targeting the microtubule cytoskeleton.

Tubulin proteomics: towards breaking the code.

Since the discovery of tubulin as the major component of microtubules over 40 years ago, its diversity of forms has raised a continuum of fundamental questions about its regulation and functions in a variety of organisms across phyla. Its high abundance in the brain or in specialized organelles such as cilia has allowed early characterization of this important target for anticancer drugs. However, it was only when matrix-assisted laser desorption ionization and electrospray ionization mass spectrometry technologies became available in the late 1980s that the full complexity of tubulin expression patterns became more obvious. This contributed in a major way to the idea that due to increasing and conserved tubulin heterogeneity during evolution, a tubulin code read by microtubule associated proteins might exist and be of functional significance. We review here the merging of recent genetic and cell biology studies with proteomics to decipher this code and illustrate some of the tubulin proteomic approaches with new data generated in our laboratories.

MALDI in-source decay of high mass protein isoforms: application to alpha- and beta-tubulin variants.

Tubulin is one of the major targets in cancer chemotherapy and the target of more than twenty percent of the cancer chemotherapic agents. The modulation of isoform content has been hypothesized as being a cause of resistance to treatment. Isoform differences lie mostly in the C-terminus part of the protein. Extensive characterization of this polypeptide region is therefore of critical importance. MALDI-TOF fragmentation of tubulin C-terminal domains was tested using synthetic peptides. Then, isotypes from HeLa cells were successfully characterized for the first time by in-source decay (ISD) fragmentation of their C-terminus coupled to a pseudo MS(3) technique named T(3)-sequencing. The fragmentation occurred in-source, preferentially generating y(n)-series ions. This approach required guanidination for the characterization of the beta(III)-tubulin C-terminus peptide. This study is, to our knowledge, the first example of reflectron in-source decay (reISD) of the C-terminus of a 50 kDa protein. This potentially occurs via a CID-like mechanism occurring in the MALDI plume. There are now new avenues for top-down characterization of important clinical biomarkers such as beta(III)-tubulin isotypes, a potential marker of drug resistance and tumor progression. This paper raises the challenge of protein isotypes characterization for early cancer detection and treatment monitoring.

Tubacin prevents neuronal migration defects and epileptic activity caused by rat Srpx2 silencing in utero

Altered development of the human cerebral cortex can cause severe malformations with often intractable focal epileptic seizures and may participate in common pathologies, notably epilepsy. This raises important conceptual and therapeutic issues. Two missense mutations in the sushi repeat-containing protein SRPX2 had been previously identified in epileptic disorders with or without structural developmental alteration of the speech cortex. In the present study, we aimed to decipher the precise developmental role of SRPX2, to have a better knowledge on the consequences of its mutations, and to start addressing therapeutic issues through the design of an appropriate animal model. Using an in utero Srpx2 silencing approach, we show that SRPX2 influences neuronal migration in the developing rat cerebral cortex. Wild-type, but not the mutant human SRPX2 proteins, rescued the neuronal migration phenotype caused by Srpx2 silencing in utero, and increased alpha-tubulin acetylation. Following in utero Srpx2 silencing, spontaneous epileptiform activity was recorded post-natally. The neuronal migration defects and the post-natal epileptic consequences were prevented early in embryos by maternal administration of tubulin deacetylase inhibitor tubacin. Hence epileptiform manifestations of developmental origin could be prevented in utero, using a transient and drug-based therapeutic protocol.

Mass spectrometry imaging is moving toward drug protein co-localization

Mass spectrometry (MS)-based technology provides label-free localization of molecules in tissue samples. Drugs, proteins, lipids and metabolites can easily be monitored in their environment. Resolution can be achieved down to the cellular level (10-20 μm) for conventional matrix-assisted laser desorption/ionization (MALDI) imaging, or even to the subcellular level for more complex technologies such as secondary ionization mass spectrometry (SIMS) imaging. One question remains: are we going to be able to investigate functional relationships between drugs and proteins and compare with localized phenomena? This review describes the various spatial levels of investigation offered by mass spectrometry imaging (MSI), and the advantages and disadvantages compared with other labeling technologies.

Gold nanoparticles prepared by laser ablation in aqueous biocompatible solutions: assessment of safety and biological identity for nanomedicine applications

Due to excellent biocompatibility, chemical stability, and promising optical properties, gold nanoparticles (Au-NPs) are the focus of research and applications in nanomedicine. Au-NPs prepared by laser ablation in aqueous biocompatible solutions present an essentially novel object that is unique in avoiding any residual toxic contaminant. This paper is conceived as the next step in development of laser-ablated Au-NPs for future in vivo applications. The aim of the study was to assess the safety, uptake, and biological behavior of laser-synthesized Au-NPs prepared in water or polymer solutions in human cell lines. Our results showed that laser ablation allows the obtaining of stable and monodisperse Au-NPs in water, polyethylene glycol, and dextran solutions. The three types of Au-NPs were internalized in human cell lines, as shown by transmission electron microscopy. Biocompatibility and safety of Au-NPs were demonstrated by analyzing cell survival and cell morphology. Furthermore, incubation of the three Au-NPs in serum-containing culture medium modified their physicochemical characteristics, such as the size and the charge. The composition of the protein corona adsorbed on Au-NPs was investigated by mass spectrometry. Regarding composition of complement C3 proteins and apolipoproteins, Au-NPs prepared in dextran solution appeared as a promising drug carrier. Altogether, our results revealed the safety of laser-ablated Au-NPs in human cell lines and support their use for theranostic applications.

MALDI imaging and in‐source decay for top‐down characterization of glioblastoma

Glioblastoma multiforme is one of the most common intracranial tumors encountered in adults. This tumor of very poor prognosis is associated with a median survival rate of approximately 14 months. One of the major issues to better understand the biology of these tumors and to optimize the therapy is to obtain the molecular structure of glioblastoma. MALDI molecular imaging enables location of molecules in tissues without labeling. However, molecular identification in situ is not an easy task. In this paper, we used MALDI imaging coupled to in‐source decay to characterize markers of this pathology. We provided MALDI molecular images up to 30 μm spatial resolution of mouse brain tissue sections. MALDI images showed the heterogeneity of the glioblastoma. In the various zones and at various development stages of the tumor, using our top‐down strategy, we identified several proteins. These proteins play key roles in tumorigenesis. Particular attention was given to the necrotic area with characterization of hemorrhage, one of the most important poor prognosis factors in glioblastoma.