ABSTRACT
Cancer gene therapy requires the design of non-viral vectors that carry genetic material and selectively deliver it with minimal toxicity. Non-viral vectors based on cationic natural polymers can form electrostatic complexes with negatively-charged polynucleotides such as microRNAs (miRNAs). Here we investigated the physicochemical/biophysical properties of chitosan-hsa-miRNA-145 (CS-miRNA) nanocomplexes and the biological responses of MCF-7 breast cancer cells cultured in vitro. Self-assembled CS-miRNA nanocomplexes were produced with a range of (+/-) charge ratios (from 0.6 to 8) using chitosans with various degrees of acetylation and molecular weight. The Z-average particle diameter of the complexes was <200 nm. The surface charge increased with increasing amount of chitosan. We observed that chitosan induces the base-stacking of miRNA in a concentration dependent manner. Surface plasmon resonance spectroscopy shows that complexes formed by low degree of acetylation chitosans are highly stable, regardless of the molecular weight. We found no evidence that these complexes were cytotoxic towards MCF-7 cells. Furthermore, CS-miRNA nanocomplexes with degree of acetylation 12% and 29% were biologically active, showing successful downregulation of target mRNA expression in MCF-7 cells. Our data, therefore, shows that CS-miRNA complexes offer a promising non-viral platform for breast cancer gene therapy.
Subject(s)
Chitosan/chemistry , MicroRNAs/metabolism , Nanostructures/chemistry , Acetylation , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/metabolism , Cell Survival/drug effects , Chitosan/toxicity , Circular Dichroism , Female , Humans , MCF-7 Cells , MicroRNAs/chemistry , Microscopy, Confocal , Particle Size , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Surface Plasmon Resonance , TransfectionABSTRACT
Chronic graft-vs-host disease (GvH), induced by injection of DBA/2 lymphocytes into (C57BL/6 x DBA/2)F1 hybrids, is a murine model for lupus nephritis, associated with a Th2-dependent polyclonal B cell activation. The development of glomerulosclerosis in this model is preceded by a glomerular influx of LFA-1+ T cells. We investigated whether exposure to bacterial superantigen would modulate the course of this autoimmune syndrome. Injection of the bacterial superantigen staphylococcal enterotoxin B (SEB) in mice has been shown to induce the activation of TcRVbeta8+ T cells. Within 2 weeks after GvH induction, mice were injected twice with 20 microg of SEB and the following parameters were examined: cytokine and Ig profile, proteinuria and renal pathology. The second SEB injection induced in GvH mice an increased release of both interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) as compared with control F1 mice. No differences were observed in IL-2 production. SEB-treated GvH mice demonstrated a delayed onset of proteinuria. Histological analysis of the kidney showed that SEB-challenged GvH mice displayed significantly more interstitial inflammation and mesangial proliferation together with more IgG2a deposits in glomeruli than non-injected GvH mice. From these results, we conclude that GvH mice are more responsive to SEB in terms of cytokine production and that bacterial infection can modulate the course of this renal disease from a membranous to a more proliferative type of nephropathy.
Subject(s)
Enterotoxins/pharmacology , Lupus Nephritis/pathology , T-Lymphocyte Subsets/drug effects , T-Lymphocyte Subsets/pathology , Animals , Chronic Disease , Cytokines/metabolism , Graft vs Host Disease/metabolism , Graft vs Host Disease/pathology , Immunoglobulin Isotypes/metabolism , Injections , Kidney/pathology , Kidney Glomerulus/metabolism , Lupus Nephritis/metabolism , Mice , Mice, Inbred C57BL , Mice, Inbred DBASubject(s)
Enterotoxins/immunology , Lymphocyte Activation , Staphylococcus aureus/immunology , Superantigens/immunology , Animals , Apoptosis , Clonal Anergy , Enterotoxins/administration & dosage , Humans , Superantigens/administration & dosage , T-Lymphocytes/immunology , T-Lymphocytes, Helper-Inducer/immunologyABSTRACT
To examine the reported heterogeneity of endothelial cells to Shiga-like toxin 1 (Stx1), the responses of human umbilical (HUVEC) and saphenous (HSVEC) vein endothelial cells to cytokines, butyrate, and toxin were compared. Untreated HSVEC were generally more susceptible than were HUVEC to Stx1; pretreatment of either cell with lipopolysaccharide, interleukin-1 beta, or tumor necrosis factor-alpha enhanced Stx1 toxicity. Dexamethasone alone increased total globotriaosylceramide (Gb3) content and toxin binding but inhibited cytokine-enhanced cytotoxicity, whereas the differentiation agent, sodium butyrate, increased both Gb3 content and cytotoxicity responses to Stx1, most prominently in HSVEC. Stx1 toxicity directly correlated with the release of von Willebrand factor from HSVEC but not from HUVEC. Thus, HUVEC and HSVEC exhibit distinctive responses to Stx1, cytokines, and butyrate. This suggests the need for caution in extrapolating from in vitro studies utilizing one endothelial cell type to in vivo events during pathogenesis of Stx-mediated thrombotic microangiopathies.
Subject(s)
Bacterial Toxins/toxicity , Butyrates/pharmacology , Cytokines/pharmacology , Cytotoxins/toxicity , Endothelium, Vascular/cytology , Bacterial Toxins/metabolism , Butyric Acid , Cells, Cultured , Cytotoxins/metabolism , Dexamethasone/pharmacology , Endothelium, Vascular/chemistry , Endothelium, Vascular/drug effects , Humans , Interleukin 1 Receptor Antagonist Protein , Interleukin-1/pharmacology , Lipopolysaccharides/pharmacology , Receptors, Cell Surface/analysis , Saphenous Vein , Shiga Toxin 1 , Sialoglycoproteins/pharmacology , Tetradecanoylphorbol Acetate/pharmacology , Trihexosylceramides/analysis , Tumor Necrosis Factor-alpha/pharmacology , Umbilical Veins , von Willebrand Factor/analysisABSTRACT
Protein S is a vitamin K-dependent plasma protein that functions as a cofactor of activated protein C (APC) in the inactivation of coagulation factors Va and VIIIa. Protein S, migrates as a doublet on reduced SDS polyacrylamide gel electrophoresis. This heterogeneity in molecular weight has been explained by limited proteolysis of protein S. Human protein S contains at Arg-49, Arg-60 and Arg-70 three potential cleavage sites. Whether cleavage occurs at all three sites is not known. To study the role of these arginine residues in human protein S, we have replaced them by leucine or isoleucine. All seven possible variants were constructed: three variants with single mutations (R49L, R60L, R70I), three variants with double mutations (R49L/R60L, R60L/R70I, R49L/R70I) and one variant with a triple mutation (R49L/R60L/R70I). On reduced SDS polyacrylamide gels the single and double variants migrate as a doublet just like the wild type protein S. The triple variant migrates as a single band at a molecular weight corresponding to the upper band of the doublet. The upper band of the single and double variants but not of the triple variant could be converted into the lower band by thrombin treatment. All variants showed cofactor activity to APC in a clotting assay. After thrombin treatment, this cofactor activity was abolished for the single (R49L, R60L, R70I) and double variants (R49L/R60L, R60L/R70I, R49L/R70I), while the triple variant (R49L/R60L/R70I) tested at several concentrations, retained its cofactor activity completely, suggesting resistance to thrombin. This shows that thrombin can cleave at all three arginine sites and that cleavage at each of these sites results in the loss of APC cofactor activity.(ABSTRACT TRUNCATED AT 250 WORDS)
