Marie-France Côté

Heparin-fibroblast growth factor-fibrin complex : in vitro and in vivo applications to collagen-based materials

Biological molecules such as fibrin and growth factors could have interesting features to design bioactive biomaterials and particularly collagen-based materials used as connective tissue replacement. Different combinations of fibroblast growth factor (FGF) and heparin complexed to fibrin were analysed. In vitro, FGF bound to matrix was rapidly, but partially released, specifically with heparin. Heparin concentrations were progressively equilibrated between matrix and medium. DNA replication of fibroblasts grown either on or within fibrin matrices was increased in the presence of both FGF and high doses of heparin incorporated in fibrin. Subcutaneous implantations of collagen sponges impregnated with composite fibrin matrices showed qualitative and quantitative tissue ingrowth within the sponges. The uncross-linked collagen of fibrin-impregnated sponges swelled after implantation. The resulting fibroblast-infiltrated tissue resembled a normal dense connective tissue that was observed particularly in the presence of high doses of heparin and FGF incorporated in fibrin.

Antiangiogenic and Antitumoral Activity of Phenyl-3-(2-Chloroethyl)Ureas A Class of Soft Alkylating Agents Disrupting Microtubules That Are Unaffected by Cell Adhesion-Mediated Drug Resistance

The development of new anticancer agents with lower toxicity, higher therapeutic index, and weaker tendency to induce resistant phenotypes in tumor cells is a continuous challenge for the scientific community. Toward that end, we showed previously that a new class of soft alkylating agents designed as phenyl-3-(2-chloroethyl)ureas (CEUs) inhibits tumor cell growth in vitro and that their efficiency is not altered by clinically relevant mechanisms of resistance such as overexpression of multidrug resistance proteins, increase in intracellular concentration of glutathione and/or glutathione S-transferase activity, alteration of topoisomerase II, and increased DNA repair. Mechanistic studies have showed recently that the cytotoxic activity of several CEUs was mainly related to the disruption of microtubules. Here, we present results supporting our assumption that 4-tert-butyl-[3-(2-chloroethyl)ureido]phenyl (tBCEU) (and its bioisosteric derivative 4-iodo-[3-(2-chloroethyl)ureido]phenyl (ICEU) are potent antimicrotubule agents both in vitro and in vivo. They covalently bind to β-tubulin, leading to a microtubule depolymerization phenotype, consequently disrupting the actin cytoskeleton and altering the nuclear morphology. Accordingly, tBCEU and ICEU also inhibited the migration and proliferation of endothelial and tumor cells in vitro in a dose-dependent manner. It is noteworthy that ICEU efficiently blocked angiogenesis and tumor growth in three distinct animal models: (a) the Matrigel plug angiogenesis assay; (b) the CT-26 tumor growth assay in mice; and (c) the chick chorioallantoic membrane tumor assay. In addition, we present evidence that CEU cytotoxicity is unaffected by additional resistance mechanisms impeding tumor response to DNA alkylating agents such as cisplatin, namely the cell adhesion mediated-drug resistance mechanism, which failed to influence the cytocidal activity of CEUs. On the basis of the apparent innocuousness of CEUs, on their ability to circumvent many classical and recently described tumor cell resistance mechanisms, and on their specific biodistribution to organs of the gastrointestinal tract, our results suggest that CEUs represent a promising new class of anticancer agents.

Porosity and biological properties of polyethylene glycol-conjugated collagen materials.

Collagen-based materials can be designed for use as scaffolds for connective tissue reconstruction. The goal of the present study was to evaluate the behavior of collagen materials as well as cell and tissue reactions after the conjugation of activated polyethylene glycols (PEGs) with collagen. It is known that proteins conjugated with PEGs exhibit a decrease in their biodegradation rate and their immunogenicity. Different concentrations and molecular weights of activated PEGs (PEG-750 and PEG-5000) were conjugated to collagen materials (films or sponges) which were then investigated by collagenase assay, fibroblast cell culture, and subcutaneous implantation. PEG-conjugated collagen sponge degradation by collagenase was delayed in comparison to untreated sponges. In culture, fibroblasts with a normal morphology reached confluency on PEG-conjugated collagen films. In vivo, the porous structure of non-modified sponges collapsed by day 15 with a few observable fibroblasts between the collagen fibers. In PEG-modified collagen sponges, the porous structure remained stable for 30 days. Cell infiltration was particularly enhanced in PEG-750-conjugated collagen sponges. In conclusion, PEGs conjugated onto collagen sponges stabilize the porous structure without deactivating the biological properties of collagen. These porous composite materials could function as a scaffold to organize tissue ingrowth.

Design, synthesis, biological evaluation, and structure-activity relationships of substituted phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates as new tubulin inhibitors mimicking combretastatin A-4.

Sixty-one phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates (PIB-SOs) and 13 of their tetrahydro-2-oxopyrimidin-1(2H)-yl analogues (PPB-SOs) were prepared and biologically evaluated. The antiproliferative activities of PIB-SOs on 16 cancer cell lines are in the nanomolar range and unaffected in cancer cells resistant to colchicine, paclitaxel, and vinblastine or overexpressing the P-glycoprotein. None of the PPB-SOs exhibit significant antiproliferative activity. PIB-SOs block the cell cycle progression in the G2/M phase and bind to the colchicine-binding site on β-tubulin leading to cytoskeleton disruption and cell death. Chick chorioallantoic membrane tumor assays show that compounds 36, 44, and 45 efficiently block angiogenesis and tumor growth at least at similar levels as combretastatin A-4 (CA-4) and exhibit low to very low toxicity on the chick embryos. PIB-SOs were subjected to CoMFA and CoMSIA analyses to establish quantitative structure–activity relationships.

Microtubule-Destabilizing Agents Induce Focal Adhesion Structure Disorganization and Anoikis in Cancer Cells

Microtubule disruption provokes cytoskeleton and cell adhesion changes whose importance for apoptosis induction remains unclear. The present study focuses on the functional and the molecular adhesion kinetics that are induced by microtubule disruption-mediated apoptosis. We showed that antimicrotubules induce a biphasic sequence of adhesion response that precedes the onset of apoptosis and focal adhesion kinase hydrolysis. Antimicrotubules first induced an increase of the cellular adhesion paralleled by the raise of focal adhesion sites and actin contractility, which was followed by a sharp decrease of cell adhesion and disorganization of focal adhesion and actin stress fibers. The latter sequence of events ends by cell rounding, detachment from the extracellular matrix, and cell death. Microtubule-disrupting agents induced a sustained paxillin phosphorylation, before the activation of apoptosis, that requires the prior activation of extracellular signal-regulated kinase and p38 but not c-Jun NH2-terminal kinase. Interestingly, integrin-linked kinase overexpression rescued the antimicrotubule-mediated loss of cell viability. Altogether, these results propound that antimicrotubule agents induce anoikis through the loss of focal adhesion structure integrity.

Inactivation of the mTORC1-Eukaryotic Translation Initiation Factor 4E Pathway Alters Stress Granule Formation

ABSTRACT Stress granules (SG) are cytoplasmic multimeric RNA bodies that form under stress conditions known to inhibit cap-dependent translation. SG contain translation initiation factors, RNA binding proteins, and signaling molecules. SG are known to inhibit apoptotic pathways, thus contributing to chemo- and radioresistance in tumor cells. However, whether stress granule formation involves oncogenic signaling pathways is currently unknown. Here, we report a novel role of the mTORC1-eukaryotic translation initiation factor 4E (eIF4E) pathway, a key regulator of cap-dependent translation initiation of oncogenic factors, in SG formation. mTORC1 specifically drives the eIF4E-mediated formation of SG through the phosphorylation of 4E-BP1, a key factor known to inhibit formation of the mTORC1-dependent eIF4E-eIF4GI interactions. Disrupting formation of SG by inactivation of mTOR with its specific inhibitor pp242 or by depletion of eIF4E or eIF4GI blocks the SG-associated antiapoptotic p21 pathway. Finally, pp242 sensitizes cancer cells to death in vitro and inhibits the growth of chemoresistant tumors in vivo. This work therefore highlights a novel role of the oncogenic mTORC1-eIF4E pathway, namely, the promotion of formation of antiapoptotic SG.

Chemical inactivators as sterilization agents for bovine collagen materials

The use of collagen as a biomedical implant raises safety issues with regard to viruses and prions. Specific chemical agents that inactivate prion infectivity could be applied to collagen implants. The physicochemical changes and the in vitro and in vivo biocompatibility of collagen treated by formic acid (FA), trifluoroacetic acid (TFA), tetrafluorethanol (TFE), and hexafluoroisopropanol (HFIP) were investigated. In addition, the effects of these treatments on nucleic acids incorporated in collagen were analyzed. The molecules of FA and, more important, of TFA remained within collagen. FA, TFA, and HFIP treatments modify the secondary structure of collagen, as shown by Fourier transform infrared spectroscopy, while TFE does not. Differential scanning calorimetry measurements showed a decrease in the denaturation temperature compared to untreated collagen. However, resistance to collagenase was modified only after HFIP treatment. In vitro, cell growth was not impaired; in vivo, implants induced a temporary inflammatory reaction that was prolonged with TFA and HFIP treatments. TFE and FA-treated collagen were thoroughly infiltrated by fibroblasts. On the other hand, FA and TFA resulted in extensive depurination of nucleic acids while HFIP and TFE did so to a lesser degree. Among the investigated chemical scrapie inactivators, FA treatment could offer a safe and biocompatible collagen-derived material for biomedical use.

Wettability of cross-linked collagenous biomaterials: In vitro study

Collagenous biomaterials can be treated by chemical and physical agents to decrease biodegradation rate. Treatments to collagen may modify surface properties and subsequently cell and platelet behaviour. Collagenous films were either uncross-linked and cross-linked by glutaraldehyde, formaldehyde or cyanamide and/or treated by a severe dehydration. Contact angles, platelet contacting assay and fibroblast morphology were investigated. After severe dehydration, wettability was diminished except for formaldehyde-cross-linked and severely dehydrated films. Glutaraldehyde-cross-linked collagen results in an increase in wettability. Platelets were similarly distributed, except on formaldehyde-cross-linked films that exhibited no platelet aggregation. Fibroblasts were in a spreading phase on most collagenous films. However, cytotoxicity was noticed on some aldehyde-cross-linked films. No direct relationship was found between contact angles and platelet-cell attachment.

New Soft Alkylating Agents with Enhanced Cytotoxicity against Cancer Cells Resistant to Chemotherapeutics and Hypoxia

Chloroethylureas (CEU) are soft alkylating agents that covalently bind to beta-tubulin (betaTAC) and affect microtubule polymerization dynamics. Herein, we report the identification of a CEU subset and its corresponding oxazolines, which induce cell growth inhibition, apoptosis, and microtubule disruption without alkylating beta-tubulin (N-betaTAC). Both betaTAC and N-betaTAC trigger the collapse of mitochondrial potential (DeltaPsi(m)) and modulate reactive oxygen species levels, following activation of intrinsic caspase-8 and caspase-9. Experiments using human fibrosarcoma HT1080 respiratory-deficient cells (rho(0)) and uncoupler of the mitochondrial respiratory chain (MRC) showed that betaTAC and N-betaTAC impaired the MRC. rho(0) cells displayed an increased sensitivity toward N-betaTAC as compared with rho(+) cells but, in contrast, were resistant to betaTAC or classic chemotherapeutics, such as paclitaxel. Oxazoline-195 (OXA-195), an N-betaTAC derivative, triggered massive swelling of isolated mitochondria. This effect was insensitive to cyclosporin A and to Bcl-2 addition. In contrast, adenine nucleotide translocator (ANT) antagonists, bongkrekic acid or atractyloside, diminished swelling induced by OXA-195. The antiproliferative activities of the N-betaTACs CEU-025 and OXA-152 were markedly decreased in the presence of atractyloside. Conversely, pretreatment with cyclosporin A enhanced growth inhibition induced by betaTAC and N-betaTAC. One of the proteins alkylated by N-betaTAC was identified as the voltage-dependent anion channel isoform-1, an ANT partner. Our results suggest that betaTAC and N-betaTAC, despite their common ability to affect the microtubule network, trigger different cytotoxic mechanisms in cancer cells. The role of mitochondria in these mechanisms and the potential of N-betaTAC as a new therapeutic approach for targeting hypoxia-resistant cells are discussed.

Bioactive collagen sponge as connective tissue substitute

Abstract Biodegradable materials such as collagen can be used as scaffold for the ingrowth of repair connective tissue. Application of interactions between extracellular matrix (ECM) molecules, growth factors and cells can be useful to design a bioactive scaffold. Studies were conducted on mice in subcutaneous implantations and in an open full-thickness wound model in pigs to investigate tissue reactions towards ECM analogues including fibrin, hyaluronic acid, fibronectin, heparin and FGF within uncrosslinked collagen sponges. In mice, subcutanous sponges impregnated with composite fibrin matrices were increasingly infiltrated by cells as a function of time. The resulting fibroblast-infiltrated tissue was close to that seen in normal connective tissue. In pigs, open wound healing was accelerated by FGF-2 and glycosaminoglycans. By 15 days, wound collagen was dense and oriented as determined by polarized light observation. Biodegradable collagen sponges in which ECM analogues have been introduced can be useful to improve cell infiltration and collagen deposition, and subsequently the healing of dermal wounds.