Doxorubicin-loaded iron oxide nanoparticles for glioblastoma therapy: A combinational approach for enhanced delivery of nanoparticles

This work is licensed under a Creative Commons Attribution 4.0 International License.Although doxorubicin (DOX) is an effective anti-cancer drug with cytotoxicity in a variety of different tumors, its effectiveness in treating glioblastoma multiforme (GBM) is constrained by insufficient penetration across the blood–brain barrier (BBB). In this study, biocompatible magnetic iron oxide nanoparticles (IONPs) stabilized with trimethoxysilylpropyl-ethylenediamine triacetic acid (EDT) were developed as a carrier of DOX for GBM chemotherapy. The DOX-loaded EDT-IONPs (DOX-EDT-IONPs) released DOX within 4 days with the capability of an accelerated release in acidic microenvironments. The DOX-loaded EDT-IONPs (DOX-EDT-IONPs) demonstrated an efficient uptake in mouse brain-derived microvessel endothelial, bEnd.3, Madin–Darby canine kidney transfected with multi-drug resistant protein 1 (MDCK-MDR1), and human U251 GBM cells. The DOX-EDT-IONPs could augment DOX’s uptake in U251 cells by 2.8-fold and significantly inhibited U251 cell proliferation. Moreover, the DOX-EDT-IONPs were found to be effective in apoptotic-induced GBM cell death (over 90%) within 48 h of treatment. Gene expression studies revealed a significant downregulation of TOP II and Ku70, crucial enzymes for DNA repair and replication, as well as MiR-155 oncogene, concomitant with an upregulation of caspase 3 and tumor suppressors i.e., p53, MEG3 and GAS5, in U251 cells upon treatment with DOX-EDT-IONPs. An in vitro MDCK-MDR1-GBM co-culture model was used to assess the BBB permeability and anti-tumor activity of the DOX-EDT-IONPs and DOX treatments. While DOX-EDT-IONP showed improved permeability of DOX across MDCK-MDR1 monolayers compared to DOX alone, cytotoxicity in U251 cells was similar in both treatment groups. Using a cadherin binding peptide (ADTC5) to transiently open tight junctions, in combination with an external magnetic field, significantly enhanced both DOX-EDT-IONP permeability and cytotoxicity in the MDCK-MDR1-GBM co-culture model. Therefore, the combination of magnetic enhanced convective diffusion and the cadherin binding peptide for transiently opening the BBB tight junctions are expected to enhance the efficacy of GBM chemotherapy using the DOX-EDT-IONPs. In general, the developed approach enables the chemotherapeutic to overcome both BBB and multidrug resistance (MDR) glioma cells while providing site-specific magnetic targeting.Canadian Institutes of Health ResearchNatural Science and Engineering Research Council—CanadaNIH R01-NS075374NIH P30-AG035982NIH T32-GM00835

Modulation of Intercellular Junctions by Cyclic-ADT Peptides as a Method to Reversibly Increase Blood-Brain Barrier Permeability

It is challenging to deliver molecules to the brain for diagnosis and treatment of brain diseases. This is primarily due to the presence of the blood-brain barrier (BBB), which restricts the entry of many molecules into the brain. In this study, cyclic ADT peptides (ADTC1, ADTC5, and ADTC6) have been shown to modify the BBB to enhance the delivery of marker molecules (e.g., 14C-mannitol, Gd-DTPA) to the brain via the paracellular pathways of the BBB. The hypothesis is that these peptides modulate cadherin interactions in the adherens junctions of the vascular endothelial cells forming the BBB to increase paracellular drug permeation. In vitro studies indicated that ADTC5 had the best profile to inhibit adherens junction resealing in MDCK cell monolayers in a concentration-dependent manner (IC50 = 0.3 mM) with a maximal response at 0.4 mM. Under the current experimental conditions, ADTC5 improved the delivery of 14C-mannitol to the brain about twofold compared to the negative control in the in situ rat brain perfusion model. Furthermore, ADTC5 peptide increased in vivo delivery of Gd-DTPA to the brain of Balb/c mice when administered intravenously (i.v.). In conclusion, ADTC5 has the potential to improve delivery of diagnostic and therapeutic agents to the brain

Regulated Expression of Chromobox Homolog 5 Revealed in Tumors of ApcMin/+ ROSA11 Gene Trap Mice

The gene-trap lacZ reporter insertion, ROSA11, in the Cbx5 mouse gene illuminates the regulatory complexity of this locus in ApcMin/+ mice. The insertion site of the β-Geo gene-trap element lies in the 24-kb intron proximal to the coding region of Cbx5. Transcript analysis indicates that two promoters for Cbx5 flank this insertion site. Heterozygotes for the insertion express lacZ widely in fetal tissues but show limited expression in adult tissues. In the intestine, strong expression is limited to proliferative zones of crypts and tumors. Homozygotes for ROSA11, found at a lower than Mendelian frequency, express reduced levels of the coding region transcript in normal tissues, using a downstream promoter. Analysis via real-time polymerase chain reaction indicates that the upstream promoter is the dominant promoter in normal epithelium and tumors. Bioinformatic analysis of the Cbx5 locus indicates that WNT and its target transcription factor MYC can establish a feedback loop that may play a role in regulating the self-renewal of the normal intestinal epithelium and its tumors

Mechanisms of simvastatin myotoxicity: The role of autophagy flux inhibition.

Statins are some of the most widely used drugs worldwide, but one of their major side effects is myotoxicity. Using mouse myoblast (C2C12) and human alveolar rhabdomyosarcoma cell lines (RH30) in both 2-dimensional (2D) and 3-dimensional (3D) cell culture, we investigated the mechanisms of simvastatin\u27s myotoxicity. We found that simvastatin significantly reduced cell viability in C2C12 cells compared to RH30 cells. However, simvastatin induced greater apoptosis in RH30 compared to C2C12 cells. Simvastatin-induced cell death is dependent on geranylgeranyl pyrophosphate (GGPP) in C2C12 cells, while in RH30 cells it is dependent on both farnesyl pyrophosphate (FPP) and GGPP. Simvastatin inhibited autophagy flux in both C2C12 and RH30 cells and inhibited lysosomal acidification in C2C12 cells, while autophagy inhibition with Bafilomycin-A1 increased simvastatin myotoxicity in both cell lines. Simvastatin induced greater cell death in RH30 cells compared to C2C12 in a 3D culture model with similar effects on autophagy flux as in 2D culture. Overall, our results suggest that simvastatin-induced myotoxicity involves both apoptosis and autophagy, where autophagy serves a pro-survival role in both cell lines. The sensitivity to simvastatin-induced myotoxicity differs between 2D and 3D culture, demonstrating that the cellular microenvironment is a critical factor in regulating simvastatin-induced cell death in myoblasts

Caspase 3 activity in isolated fetal rat lung fibroblasts and rat periodontal ligament fibroblasts: cigarette smoke induced alterations

Background Cigarette smoking is the leading cause of preventable death and has been implicated in pathogenesis of pulmonary, oral and systemic diseases. Smoking during pregnancy is a risk factor for the developing fetus and may be a major cause of infant mortality. Moreover, the oral cavity, and all cells within are the first to be exposed to cigarette smoke and may be a possible source for the spread of toxins to other organs of the body. Fibroblasts in general are morphologically heterogeneous connective tissue cells with diverse functions. Apoptosis or programmed cell death is a crucial process during embryogenesis and for the maintenance of homeostasis throughout life. Deregulation of apoptosis has been implicated in abnormal lung development in the fetus and disease progression in adults. Caspases are proteases which belong to the family of cysteine aspartic acid proteases and are key components for downstream amplification of intracellular apoptotic signals. Of 14 known caspases, caspase-3 is the key executioner of apoptosis. In the present study we explored the hypothesis that cigarette smoke (CS) extract activates caspase-3 in two types of fibroblasts, both of which would be exposed directly to cigarette smoke, isolated fetal rat lung fibroblasts and adult rat periodontal ligament (PDL) fibroblasts. Methods Isolated fetal rat lung fibroblasts and adult PDLs were used. Cells were exposed to different concentrations of CS for 60 min. Caspase-3 activity and its inhibition by Z-VAD-fmk were measured by caspase-3 fluorometric assay. The effect of CSE on cellular viability was measured using the MTT formazan assay. Caspase-3 expression was detected by western blot analysis and cellular localization of caspase-3 was determined by immunofluorescence using fluorescence microscopy. Results It was observed in fetal rat lung fibroblast cells that CSE extract significantly (p<0.05) increased caspase-3 activity and decrease cell proliferation. However, no significant changes in activity or viability were observed in PDLs. Conclusions This indicates CS activates caspase-3 the key regulatory point in apoptosis in fetal rat lung fibroblast cells suggesting that smoking during pregnancy may alter the developmental program of fetal lung, jeopardizing the establishment of critical cellular mechanisms necessary to expedite pulmonary maturation at birth.of critical cellular mechanisms necessary to expedite pulmonary maturation at birth

Analysis of neoplastic changes in mouse lung using Fourier-transform infrared microspectroscopy

Methods based on infrared microspectroscopy were explored as a means to distinguish normal from neoplastic lung tissue. Mice were exposed to urethane, a known environmental carcinogen. After 3\u20138 months, lungs were removed, snap-frozen, sectioned and analyzed by standard histological methods and by infrared microspectroscopy. Neoplasms were readily observed in mice treated with urethane. Ultra-structurally, the neoplasms were composed entirely of type II alveolar cells displaying intracellular lamellar bodies. A fusion of two spectral pre-processing techniques, optimal region selection and linear discriminant analysis (LDA), was used to search for infrared spectral signatures distinguishing normal from neoplastic tissue. These techniques showed clear and reproducible differences between the complex spectra of these tissue types, suggesting that infrared microspectroscopy in conjunction with spectral processing technology may be useful to reveal subtle spectral differences occurring following induction of neoplastic changes and to interpret their biochemical origins.Peer reviewed: YesNRC publication: Ye

Clinical modalities for the diagnosis, characterization and detection of bone metastases: Bone Metastatis and Molecular Mechanisms

NRC publication: Ye