RESUMO
Triple negative breast cancers (TNBC) are aggressive malignancies for which chemotherapy is the only treatment option. Many TNBC acquire chemotherapy resistance, notably docetaxel, which has been associated with the overexpression of transcription factors (TFs), such as ENGRAILED1 (EN1). Here, we have developed a tumor delivery system for docetaxel-PGMA-PAA-nanoparticles and interference peptides designed to specifically inhibit EN1 (EN1-iPeps). To promote tumor specific targeting, we functionalized these nanoparticles with EN1-iPeps engineered with RGD sequences. We found that these peptides reduce cell viability and induce apoptosis in TNBC cells with negligible effects on normal cells (EN1-). Moreover, EN1-RGD-iPeps-mediated nanoparticle internalization into breast cancer cells was via integrins and intravenous injection of this nanoformulation increased tumor accumulation. Furthermore, docetaxel nanoparticles functionalized with EN1-RGD-iPeps significantly reduced TNBC growth both in vitro and in vivo without showing toxicity. Our results suggest that this targeted nanoformulation represents a new and safe therapeutic approach for chemoresistant TNBCs.
Assuntos
Docetaxel/uso terapêutico , Proteínas de Homeodomínio/metabolismo , Nanopartículas/química , Oligopeptídeos/química , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Docetaxel/farmacologia , Endocitose/efeitos dos fármacos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Células NIH 3T3 , Polímeros/química , Distribuição Tecidual/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
BACKGROUND: Following injury to the central nervous system, increased microglia, secretion of pro- and anti-inflammatory cytokines, and altered blood-brain barrier permeability, a hallmark of degeneration, are observed at and immediately adjacent to the injury site. However, few studies investigate how regions remote from the primary injury could also suffer from inflammation and secondary degeneration. METHODS: Adult female Piebald-Viral-Glaxo (PVG) rats underwent partial transection of the right optic nerve, with normal, age-matched, unoperated animals as controls. Perfusion-fixed brains and right optic nerves were harvested for immunohistochemical assessment of inflammatory markers and blood-brain barrier integrity; fresh-frozen brains were used for multiplex cytokine analysis. RESULTS: Immediately ventral to the optic nerve injury, immunointensity of both the pro-inflammatory biomarker inducible nitric oxide synthase (iNOS) and the anti-inflammatory biomarker arginase-1 (Arg1) increased at 7 days post-injury, with colocalization of iNOS and Arg1 immunoreactivity within individual cells. CD11b+ and CD45+ cells were increased 7 days post-injury, with altered BBB permeability still evident at this time. In the lower and middle optic tract and superior colliculus, IBA1+ resident microglia were first increased at 3 days; ED1+ and CD11b+ cells were first increased in the middle and upper tract and superior colliculus 7 days post-injury. Increased fibrinogen immunoreactivity indicative of altered BBB permeability was first observed in the contralateral upper tract at 3 days and middle tract at 7 days post-injury. Multiplex cytokine analysis of brain homogenates indicated significant increases in the pro-inflammatory cytokines, IL-2 and TNFα, and anti-inflammatory cytokine IL-10 1 day post-injury, decreasing to control levels at 3 days for TNFα and 7 days for IL-2. IL-10 was significantly elevated at 1 and 7 days post-injury with a dip at 3 days post-injury. CONCLUSIONS: Partial injury to the optic nerve induces a complex remote inflammatory response, characterized by rapidly increased pro- and anti-inflammatory cytokines in brain homogenates, increased numbers of IBA1+ cells throughout the visual pathways, and increased CD11b+ and ED1+ inflammatory cells, particularly towards the synaptic terminals. BBB permeability can increase prior to inflammatory cell infiltration, dependent on the brain region.
Assuntos
Barreira Hematoencefálica/patologia , Citocinas/metabolismo , Encefalite/etiologia , Traumatismos do Nervo Óptico/complicações , Traumatismos do Nervo Óptico/patologia , Vias Visuais/patologia , Análise de Variância , Animais , Antígenos CD/metabolismo , Barreira Hematoencefálica/fisiopatologia , Proteínas de Ligação ao Cálcio/metabolismo , Modelos Animais de Doenças , Ectodisplasinas/metabolismo , Encefalite/patologia , Feminino , Fibrinogênio/metabolismo , Lateralidade Funcional , Macrófagos/patologia , Proteínas dos Microfilamentos/metabolismo , Microglia/patologia , Óxido Nítrico Sintase Tipo II/metabolismo , Nervo Óptico/patologia , Ratos , Fatores de Tempo , Vias Visuais/metabolismoRESUMO
Introduction: Immune checkpoint inhibitors (ICIs) have revolutionized cancer therapeutics. However, immune-related adverse events (irAEs) increase morbidity and mortality and thereby limit therapeutic utility. The real-world incidence of the entire spectrum of pulmonary irAEs has not been systematically described. The objective of this study is to assess the risk of developing pulmonary irAEs (pneumonitis, pleural events [i.e., effusion and pleurisy], exacerbations of airway disease [i.e., bronchitis and bronchiectasis], and sarcoidosis) with exposure to five commonly used ICIs: nivolumab, pembrolizumab, durvalumab, avelumab, and atezolizumab. Methods: We conducted a retrospective review of the Food and Drug Administration Adverse Events Reporting System (FAERS) pharmacovigilance database. We collected data from 2012 to 2021 to assess the risk of pulmonary irAEs and performed a disproportionality analysis using Open-Vigil, a software package used for analysis of pharmacovigilance data, to calculate reporting odds ratios (RORs). We used 95% CIs to evaluate the precision of RORs. An ROR greater than 1 and the upper limit of the 95% CI indicated statistical significance. Results: A total of 17,273,403 events were reported in FAERS between 2012 and 2021. Of these, 88,099 (0.5%) were attributed to the PD-1 (programmed cell death protein 1) inhibitors and 21,905 (0.1%) to PD-L1 (programmed death ligand 1) inhibitors of interest. The most common indication for using the ICIs of interest was lung cancer: a total of 2832 (46.70%) for the PD-1 inhibitors and 1311 (70.9%) for the PD-L1 inhibitors. In the anti-PD-1 group, 2342 (38.6%) patients were hospitalized, and 1962 (32.4%) patients died from the lung adverse event. In the PD-L1 group, 744 (40.3%) patients were hospitalized, and 520 (28.1%) patients died from the event. Nivolumab resulted in the highest statistically significant risk (ROR, 10.5; 95% CI, 10.1-10.9) for pneumonitis. Avelumab had a lesser risk for pneumonitis (ROR, 0.2; 95% CI, 0.2-0.3). The risk for pleural events was highest with nivolumab (ROR, 3.6; 95% CI, 3.4-3.9), followed by pembrolizumab (ROR, 1.8; 95% CI; 1.6-2.0) (p < 0.001), with the lowest risks from durvalumab, atezolizumab, and avelumab. For ICI-related sarcoidosis, the risk was most significant with pembrolizumab (ROR, 3.6; 95% CI, 2.8-4.7), followed by nivolumab (ROR, 2.5; 95% CI, 1.9-3.5) (p < 0.001). The RORs for all five ICIs were less than 1 for exacerbations of airway diseases as compared with other drugs. Conclusion: Using a pharmacovigilance database, we found an increased risk of multiple pulmonary irAEs after ICI therapy, particularly with PD-1 inhibitors. Further work is needed to investigate the incidence of pulmonary irAEs other than pneumonitis.
RESUMO
As millimetre wave (MMW) frequencies of the electromagnetic spectrum are increasingly adopted in modern technologies such as mobile communications and networking, characterising the biological effects is critical in determining safe exposure levels. We study the exposure of primary human dermal fibroblasts to MMWs, finding MMWs trigger genomic and transcriptomic alterations. In particular, repeated 60 GHz, 2.6 mW cm-2, 46.8 J cm-2 d-1 MMW doses induce a unique physiological response after 2 and 4 days exposure. We show that high dose MMWs induce simultaneous non-thermal alterations to the transcriptome and DNA structural dynamics, including formation of G-quadruplex and i-motif secondary structures, but not DNA damage.
RESUMO
The evolution of gaseous products is a feature common to several electrochemical processes, often resulting in bubbles adhering to the electrode's surface. Adherent bubbles reduce the electrode active area, and are therefore generally treated as electrochemically inert entities. Here, we show that this general assumption does not hold for gas bubbles masking anodes operating in water. By means of imaging electrochemiluminescent systems, and by studying the anisotropy of polymer growth around bubbles, we demonstrate that gas cavities adhering to an electrode surface initiate the oxidation of water-soluble species more effectively than electrode areas free of bubbles. The corona of a bubble accumulates hydroxide anions, unbalanced by cations, a phenomenon which causes the oxidation of hydroxide ions to hydroxyl radicals to occur at potentials at least 0.7 V below redox tabled values. The downhill shift of the hydroxide oxidation at the corona of the bubble is likely to be a general mechanism involved in the initiation of heterogeneous electrochemical reactions in water, and could be harnessed in chemical synthesis.
RESUMO
An aromatic triarylpyridine chromophore promotes pi-stacking interactions with the terminal G-tetrad in quadruplex DNA, stabilizing the structure and presenting a pathway towards cancer treatment by inhibition of telomerase. An interesting parent compound in this class is the dimethylamino functionalised 4'-aryl-2,6-bis(4-aminophenyl)pyridine. However, access to this compound using traditional batch synthetic methodology is limited, due to thermodynamic and kinetic constraints. A novel approach to the synthesis of this compound has been developed, involving dynamic thin films, overcoming a series of competing reactions, effectively controlling chemical reactivity and selectivity.
Assuntos
DNA/química , DNA/metabolismo , Quadruplex G , Microfluídica/métodos , Piridinas/síntese química , Piridinas/metabolismo , Sequência de Bases , DNA/genética , Transferência Ressonante de Energia de Fluorescência , Ligantes , Microfluídica/instrumentação , Modelos Moleculares , Conformação Molecular , Piridinas/químicaRESUMO
The potential impact of CRISPR/Cas9, TALE, and zinc finger technology is immense, both with respect to their use as tools for understanding the roles and functions of the genomic elements and epigenome modifications in an endogenous context and as new methods for treatment of diseases. Application of such technologies has drawn attention, however, to the prevailing lack of effective delivery methods. Promising viral and non-viral methods both currently fall short when the efficient delivery of large plasmids or multiple plasmids is required. Therefore, the use of TALE and CRISPR platforms has been severely limited in applications where selection methods to increase the relative proportion of treated cells are not applicable, and it represents a significant bottleneck in the further application of these tools as therapeutics.The protocol presented here describes the synthesis of a dendronized polymer as a highly efficient and nontoxic transfection agent. Furthermore, the optimization of the polymer as a co-transfection reagent for large and multiple plasmids in cell lines is described, in addition to general considerations for co-transfection experiments. Usage of this method has allowed for significantly improved large plasmid co-transfection efficiency over Lipofectamine 2000 in multiple cell lines, allowing an improved delivery of CRISPR/dCas9 and TALE systems.
Assuntos
Química Click/métodos , Dendrímeros/síntese química , Transfecção/métodos , Animais , Azidas/síntese química , Azidas/química , Sistemas CRISPR-Cas , Linhagem Celular , Dendrímeros/química , Halogenação , Humanos , Transfecção/economiaRESUMO
Intracellular delivery of M6P/IGFII receptor inhibitors exhibits better efficacy than extracellular inhibitors to regulate TGFß1 mediated upregulation of profibrotic marker, collagen I.
RESUMO
Glutamate excitotoxicity contributes to damage following injury to the central nervous system via mechanisms including changes in the expression of receptors, calcium overload, oxidative stress and cell death. Excitotoxicity is triggered by glutamate binding to receptors, including calcium permeable AMPA receptors, which can be upregulated under injury conditions. However, the transcriptional response of AMPA receptor regulatory proteins to excitotoxic conditions is unknown. Here, we use real-time quantitative PCR (RT-qPCR), to determine the effect of prolonged glutamate excitotoxicity on the expression of mRNA encoding for GluR1 and AMPA receptor regulatory proteins in dissociated rat retinal cultures that include neuronal (retinal ganglion cell (RGCs)) and glial (Müller) cell populations. mRNA levels of GluR1 and regulators of the GluR1 subunit of AMPA receptors, including Sap97, Cnih2 and Cnih3, decreased following 6, 24 and 48 h incubation with 5 mM glutamate: related regulators not associated with GluR1 were unaffected. In contrast, GluR1 protein, assessed immunohistochemically, was increased in both RGCs and Müller cells after 24 h glutamate exposure: western blotting analysis was inconclusive. Reductions in mRNA of GluR1 and associated regulators occurred prior to cell death, which was first detected at 24 h, and substantial by 48 h. Exposure to glutamate acutely increased the intracellular calcium concentration in the cultures and by 24 h, reactive oxygen species were increased. Our data suggest a negative feedback mechanism in retinal cells, that down-regulates transcription of genes encoding GluR1 regulatory proteins in response to glutamate exposure. Despite this mechanism, GluR1 protein levels remain increased, and are associated with increased reactive species and cell death. Therapeutic strategies targeting calcium permeable AMPA receptors should take into account that increases in GluR1 protein are not necessarily associated with increases in associated mRNA levels over time.
Assuntos
Regulação para Baixo/efeitos dos fármacos , Proteínas do Olho/metabolismo , Ácido Glutâmico/farmacologia , Receptores de AMPA/biossíntese , Retina/metabolismo , Doenças Retinianas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Membrana/metabolismo , Ratos , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Retina/patologia , Doenças Retinianas/patologiaRESUMO
A significant change of surface features of malignant cervical epithelial cells compared to normal cells has been previously reported. Here, we are studying the question at which progressive stage leading to cervical cancer the surface alteration happens. A non-traditional method to identify malignant cervical epithelial cells in vitro, which is based on physical (in contrast to specific biochemical) labelling of cells with fluorescent silica micron-size beads, is used here to examine cells at progressive stages leading to cervical cancer which include normal epithelial cells, cells infected with human papillomavirus type-16 (HPV-16), cells immortalized by HPV-16, and carcinoma cells. The study shows a statistically significant (at p < 0.01) difference between both immortal and cancer cells and a group consisting of normal and infected. There is no significant difference between normal and infected cells. Immortal cells demonstrate the signal which is closer to cancer cells than to either normal or infected cells. This implies that the cell surface, surface cellular brush changes substantially when cells become immortal. Physical labeling of the cell surface represents a substantial departure from the traditional biochemical labeling methods. The results presented show the potential significance of physical properties of the cell surface for development of clinical methods for early detection of cervical cancer, even at the stage of immortalized, premalignant cells.
Assuntos
Transformação Celular Viral , Células Epiteliais/patologia , Papillomavirus Humano 16 , Infecções por Papillomavirus/patologia , Neoplasias do Colo do Útero/patologia , Adesão Celular , Células Epiteliais/virologia , Feminino , Corantes Fluorescentes/química , Humanos , Microscopia de Fluorescência , Microesferas , Infecções por Papillomavirus/virologia , Rodaminas/química , Coloração e Rotulagem , Células Tumorais Cultivadas , Neoplasias do Colo do Útero/virologiaRESUMO
RADA16 self-assembling peptide nanofiber scaffolds (SAPNSs) have been shown to have positive effects on neural regeneration following injury to the central nervous system in vivo, but mechanisms are unclear. Here we show that RADA16 SAPNSs form scaffolds of increasing fiber density with increasing peptide concentration which in turn has a concentration-dependent effect on neurons and astrocytes in mixed retinal cultures. Importantly, we report that the final nanoscale fiber architecture is an important factor to consider in designing scaffolds to promote regeneration in the central nervous system.