Design of PEGylated Three Ligands Silica Nanoparticles for Multi-Receptor Targeting.

The synthesis of silica nanoparticles (SiNPs) decorated on their surface with a range of various elements (e.g., ligands, drugs, fluorophores, vectors, etc.) in a controlled ratio remains a big challenge. We have previously developed an efficient strategy to obtain in one-step, well-defined multifunctional fluorescent SiNPs displaying fluorophores and two peptides ligands as targeting elements, allowing selective detection of cancer cells. In this paper, we demonstrate that additional level of controlled multifunctionality can be achieved, getting even closer to the original concept of "magic bullet", using solely sol-gel chemistry to achieve conjugation of PEG chains for stealth, along with three different ligands. In addition, we have answered the recurrent question of the surface ungrafting by investigating the stability of different siloxane linkages with the ERETIC Method (Electronic Reference to Access In Vivo Concentrations) by 19F NMR quantification. We also compared the efficiency of the hybrid silylated fluorophore covalent linkage in the core of the SiNP to conventional methods. Finally, the tumor-cell-targeting efficiency of these multi-ligand NPs on human endothelial cells (HUVEC or HDMEC) and mixed spheroids of human melanoma cells and HUVEC displaying different types of receptors were evaluated in vitro.

The pyrrolopyrimidine colchicine-binding site agent PP-13 reduces the metastatic dissemination of invasive cancer cells in vitro and in vivo.

Standard chemotherapies that interfere with microtubule dynamics are a chemotherapeutic option used for the patients with advanced malignancies that invariably relapse after targeted therapies. However, major efforts are needed to reduce their toxicity, optimize their efficacy, and reduce cancer chemoresistance to these agents. We previously identified a pyrrolo[2,3d]pyrimidine-based microtubule-depolymerizing agent (PP-13) that binds to the colchicine site of ß-tubulin and exhibits anticancer properties in solid human cancer cells, including chemoresistant subtypes. Here, we investigated the therapeutic potential of PP-13 in vitro and in vivo. PP-13 induced a mitotic blockade and apoptosis in several cancer cells cultured in two-dimensions or three-dimensions spheroids, in conjunction with reduced cell proliferation. Capillary-like tube formation assays using HUVECs showed that PP-13 displayed antiangiogenic properties. It also inhibited cancer cell motility and invasion, in in vitro wound-healing and transwell migration assays. Low concentration PP-13 (130 nmol.L-1) treatment significantly reduced the metastatic invasiveness of human cancer cells engrafts on chicken chorioallantoic membrane. In nude mice, 0.5 or 1 mg.kg-1 PP-13 intraperitoneally administered three-times a week reduced the sizes of paclitaxel-refractory orthotopic breast tumors, delayed the progression of metastasis, and decreased the global metastatic load compared to 0.5 mg.kg-1 paclitaxel or vehicle alone. PP-13 did not show any apparent early adverse effect in vivo. These data suggest that PP-13 is a promising alternative to standard chemotherapy in antimitotic drug-refractory tumors, especially through its impact on metastasis.

Anti-tumor efficacy of hyaluronan-based nanoparticles for the co-delivery of drugs in lung cancer.

Combinations of therapeutic agents could synergistically enhance the response of lung cancer cells. Co-delivery systems capable of transporting chemotherapeutics with different physicochemical properties and with the simultaneous release of drugs remain elusive. Here, we assess the ability of nanoparticles of 30-nm diameter obtained from the self-assembly of hyaluronan-based copolymer targeting CD44 receptors to encapsulate both gefitinib and vorinostat for effective combinational lung cancer treatment. Drug loading was performed by nanoprecipitation. Drug release experiments showed a slow release of both drugs after 5 days. Using two- and three-dimensional lung adenocarcinoma cell cultures, we observed that the nanoparticles were mostly found at the periphery of the CD44-expressing spheroids. These drug-loaded nanoparticles were as cytotoxic as free drugs in the two- and three-dimensional systems and toxicity was due to apoptosis induction. In mouse models, intravenous injection of hyaluronan-based nanoparticles showed a selective delivery to subcutaneous CD44-overexpressing tumors, despite a significant liver capture. In addition, the systemic toxicity of the free drugs was reduced by their co-delivery using the nanoparticles. Finally, intrapulmonary administration of drug-loaded nanoparticles, to avoid a possible hepatic toxicity due to their accumulation in the liver, showed a stronger inhibition of orthotopic lung tumor growth compared to free drugs. In conclusion, hyaluronan-based nanoparticles provide active targeting partially mediated by CD44, less-toxic drug release and improved antitumor efficiency.

Unsuccessful mitosis in multicellular tumour spheroids.

Multicellular spheroids are very attractive models in oncology because they mimic the 3D organization of the tumour cells with their microenvironment. We show here using 3 different cell types (mammary TSA/pc, embryonic kidney Hek293 and cervical cancer HeLa), that when the cells are growing as spheroids the frequency of binucleated cells is augmented as occurs in some human tumours.We therefore describe mitosis in multicellular spheroids by following mitotic markers and by time-lapse experiments. Chromosomes alignment appears to be correct on the metaphasic plate and the passenger complex is well localized on centromere. Moreover aurora kinases are fully active and histone H3 is phosphorylated on Ser 10. Consequently, the mitotic spindle checkpoint is satisfied and, anaphase proceeds as illustrated by the transfer of survivin on the spindle and by the segregation of the two lots of chromosomes. However, the segregation plane is not well defined and oscillations of the dividing cells are observed. Finally, cytokinesis fails and the absence of separation of the two daughter cells gives rise to binucleated cells.Division orientation is specified during interphase and persists throughout mitosis. Our data indicate that the cancer cells, in multicellular spheroids, lose their ability to regulate their orientation, a feature commonly encountered in tumours.Moreover, multicellular spheroid expansion is still sensitive to mitotic drugs as pactlitaxel and aurora kinase inhibitors. The spheroids thus represent a highly relevant model for studying drug efficiency in tumours.

Discovery of benzo[e]pyridoindolones as kinase inhibitors that disrupt mitosis exit while erasing AMPK-Thr172 phosphorylation on the spindle.

Aurora kinases play an essential role in mitotic progression and are attractive targets in cancer therapy. The first generation of benzo[e]pyridoindole exhibited powerful aurora kinase inhibition but their low solubility limited further development. Grafting a pyperidine-ethoxy group gives rise to a hydrosoluble inhibitor: compound C5M.C5M could efficiently inhibit the proliferation of cells from different origins. C5M prevented cell cycling, induced a strong mitotic arrest then, cells became polyploid and finally died. C5M did not impair the spindle checkpoint, the separation of the sister chromatids and the transfer of aurora B on the mid-zone. C5M prevented histone H3 phosphorylation at mitotic entry and erased AMPK-Thr172 phosphorylation in late mitosis. With this unique profile of inhibition, C5M could be useful for understanding the role of phospho-Thr172-AMPK in abscission and the relationship between the chromosomal complex and the energy sensing machinery.C5M is a multikinase inhibitor with interesting preclinical characteristics: high hydro-solubility and a good stability in plasma. A single dose prevents the expansion of multicellular spheroids. C5M can safely be injected to mice and reduces significantly the development of xenograft. The next step will be to define the protocol of treatment and the cancer therapeutic field of this new anti-proliferative drug.

Snake venoms as a source of compounds modulating sperm physiology: Secreted phospholipases A2 from Oxyuranus scutellatus scutellatus impact sperm motility, acrosome reaction and in vitro fertilization in mice.

The goal of this study was to identify new compounds from venoms able to modulate sperm physiology and more particularly sperm motility. For this purpose, we screened the effects of 16 snake venoms cleared of molecules higher than 15 kDa on sperm motility. Venoms rich in neurotoxins like those from Oxyuranus scutellatus scutellatus or Daboia russelii, were highly potent inhibitors of sperm motility. In contrast, venoms rich in myotoxins like those from Echis carinatus, Bothrops alternatus and Macrovipera lebetina, were inactive. From the main pharmacologically-active fraction of the Taipan snake O. scutellatus s., a proteomic approach allowed us to identify 16 different proteins, among which OS1 and OS2, two secreted phospholipases A2 (sPLA(2)). Purified OS1 and OS2 mimicked the inhibitory effect on sperm motility and were likely responsible for the inhibitory effect of the active fraction. OS1 and OS2 triggered sperm acrosome reaction and induced lipid rearrangements of the plasma membrane. The catalytic activity of OS2 was required to modulate sperm physiology since catalytically inactive mutants had no effect. Finally, sperm treated with OS2 were less competent than control sperm to initiate in vitro normal embryo development. This is the first report characterizing sPLA(2) toxins that modulate in vitro sperm physiology.

Two-dimensional fluorescence difference gel electrophoresis analysis of spermatogenesis in the rat.

The molecular mechanisms underlying normal and pathological spermatogenesis remain poorly understood. We compared protein concentrations in different germ cell types to identify those proteins specifically or preferentially expressed at each stage of rat spermatogenesis. Crude cytosolic protein extracts and reversed-phase HPLC prefractionated cytosolic extracts from spermatogonia, pachytene spermatocytes, and early spermatids were subjected to two-dimensional difference gel electrophoresis (2-D DIGE). By comparing gels and carrying out statistical analyses, we were able to identify 1274 protein spots with relative abundances differing significantly between the three cell types. We found that 265 of these spots displaying highly differential expression (ratio > or = 2.5 between two cell types), identified by mass fingerprinting, corresponded to 123 nonredundant proteins. The proteins clustered into three clades, corresponding to mitotic, meiotic, and post-meiotic cell types. The differentially expressed proteins identified by 2-D DIGE were confirmed and validated by western blotting and immunohistochemistry, in the few cases in which antibodies were available. 2-D DIGE appears a relevant proteomics approach for studying rat germ cell differentiation, allowing the establishment of the precise expression profiles for a relatively large number of proteins during normal spermatogenesis.