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The important role of unconventional myosin VI (MVI) in skeletal and cardiac muscle has been recently postulated (Karolczak et al. in Histochem Cell Biol 139:873-885, 2013). Here, we addressed for the first time a role for this unique myosin motor in myogenic cells as well as during their differentiation into myotubes. During myoblast differentiation, the isoform expression pattern of MVI and its subcellular localization underwent changes. In undifferentiated myoblasts, MVI-stained puncti were seen throughout the cytoplasm and were in close proximity to actin filaments, Golgi apparatus, vinculin-, and talin-rich focal adhesion as well as endoplasmic reticulum. Colocalization of MVI with endoplasmic reticulum was enhanced during myotube formation, and differentiation-dependent association was also seen in sarcoplasmic reticulum of neonatal rat cardiomyocytes (NRCs). Moreover, we observed enrichment of MVI in myotube regions containing acetylcholine receptor-rich clusters, suggesting its involvement in the organization of the muscle postsynaptic machinery. Overexpression of the H246R MVI mutant (associated with hypertrophic cardiomyopathy) in myoblasts and NRCs caused the formation of abnormally large intracellular vesicles. MVI knockdown caused changes in myoblast morphology and inhibition of their migration. On the subcellular level, MVI-depleted myoblasts exhibited aberrations in the organization of actin cytoskeleton and adhesive structures as well as in integrity of Golgi apparatus and endoplasmic reticulum. Also, MVI depletion or overexpression of H246R mutant caused the formation of significantly wider or aberrant myotubes, respectively, indicative of involvement of MVI in myoblast differentiation. The presented results suggest an important role for MVI in myogenic cells and possibly in myoblast differentiation.
Assuntos
Desenvolvimento Muscular , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/fisiologia , Miócitos Cardíacos/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Animais , Adesão Celular , Diferenciação Celular , Linhagem Celular , Movimento Celular , Forma Celular , Citoplasma/metabolismo , Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/ultraestrutura , Camundongos , Mioblastos/ultraestrutura , Miócitos Cardíacos/ultraestrutura , Cadeias Pesadas de Miosina/química , Ratos , Retículo Sarcoplasmático/metabolismoRESUMO
Silk is a biocompatible and biodegradable material that enables the formation of various morphological forms, including nanospheres. The functionalization of bioengineered silk makes it possible to produce particles with specific properties. In addition to tumor cells, the tumor microenvironment (TME) includes stromal, immune, endothelial cells, signaling molecules, and the extracellular matrix (ECM). Matrix metalloproteinases (MMPs) are overexpressed in TME. We investigated bioengineered spider silks functionalized with MMP-responsive peptides to obtain targeted drug release from spheres within TME. Soluble silks MS12.2MS1, MS12.9MS1, and MS22.9MS2 and the corresponding silk spheres carrying MMP-2 or MMP-2/9 responsive peptides were produced, loaded with doxorubicin (Dox), and analyzed for their susceptibility to MMP-2/9 digestion. Although all variants of functionalized silks and spheres were specifically degraded by MMP-2/9, the MS22.9MS2 nanospheres showed the highest levels of degradation and release of Dox after enzyme treatment. Moreover, functionalized spheres were degraded in the presence of cancer cells releasing MMP-2/9. In the 2D and 3D spheroid cancer models, the MMP-2/9-responsive substrate was degraded and released from spheres when loaded into MS22.9MS2 particles but not into the control MS2 spheres. The present study demonstrated that a silk-based MMP-responsive delivery system could be used for controlled drug release within the tumor microenvironment.
Assuntos
Preparações de Ação Retardada , Doxorrubicina , Liberação Controlada de Fármacos , Metaloproteinase 2 da Matriz , Seda , Microambiente Tumoral , Microambiente Tumoral/efeitos dos fármacos , Doxorrubicina/farmacologia , Doxorrubicina/química , Humanos , Seda/química , Metaloproteinase 2 da Matriz/metabolismo , Preparações de Ação Retardada/farmacologia , Metaloproteinase 9 da Matriz/metabolismo , Linhagem Celular Tumoral , Metaloproteinases da Matriz/metabolismo , Portadores de Fármacos/química , AnimaisRESUMO
High expression and phosphorylation of signal transducer and transcription activator 3 (STAT3) are correlated with progression and poor prognosis in various types of cancer. The constitutive activation of STAT3 in cancer affects processes such as cell proliferation, apoptosis, metastasis, angiogenesis, and drug resistance. The importance of STAT3 in cancer makes it a potential therapeutic target. Various methods of directly and indirectly blocking STAT3 activity at different steps of the STAT3 pathway have been investigated. However, the outcome has been limited, mainly by the number of upstream proteins that can reactivate STAT3 or the relatively low specificity of the inhibitors. A new branch of molecules with significant therapeutic potential has emerged thanks to recent developments in the regulatory function of non-coding nucleic acids. Oligonucleotide-based therapeutics can silence target transcripts or edit genes, leading to the modification of gene expression profiles, causing cell death or restoring cell function. Moreover, they can reach untreatable targets, such as transcription factors. This review briefly describes oligonucleotide-based therapeutics that found application to target STAT3 activity in cancer. Additionally, this review comprehensively summarizes how the inhibition of STAT3 activity by nucleic acid-based therapeutics such as siRNA, shRNA, ASO, and ODN-decoy affected the therapy of different types of cancer in preclinical and clinical studies. Moreover, due to some limitations of oligonucleotide-based therapeutics, the importance of carriers that can deliver nucleic acid molecules to affect the STAT3 in cancer cells and cells of the tumor microenvironment (TME) was pointed out. Combining a high specificity of oligonucleotide-based therapeutics toward their targets and functionalized nanoparticles toward cell type can generate very efficient formulations.
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Hereditary familial adenomatous polyposis (FAP) in humans significantly increases the risk of development of colorectal cancer (CRC). Germline mutations in the APC (adenomatous polyposis coli) gene are responsible for FAP. Despite having the same causative mutation, the severity of the disease differs from patient to patient. The porcine FAP model carrying a truncating APC1311 mutation, orthologous to the dominant human mutation that leads to severe form of the disease (APC1309), mirrors the severity of polyposis. Earlier RNAseq studies have revealed the differential expression of WISP1 and CSF1R in samples derived from low-grade (LG-IEN) and more advanced high-grade (HG-IEN) colon polyps of APC1311/+ pigs. The grade of dysplasia was correlated with the severity of polyposis in APC1311/+ pigs characterized by a low (LP) and high (HP) numbers of polyps. The goal of this work was to find DNA variants that regulate the expression of CSF1R and WISP1 in LP and HP pigs. In total, 32 and 36 polymorphisms in CSF1R and WISP1 were found, respectively. Of these, the genotype frequency of four silent SNPs in the coding region of WISP1 differed significantly between LP and HP lines. In silico analysis revealed an elevated minimum free energy (MFE) for three of these SNPs, suggesting their role in mRNA structure stability. Furthermore, four polymorphisms in the promoter region of CSF1R, cosegregating as a common haplotype, were associated with polyp number in APC1311/+ pigs. A secreted alkaline phosphatase (SEAP) assay showed, however, that these variants have no direct effect on the activity of the CSF1R promoter. Concluding, our study identified polymorphisms in CSF1R and WISP1 that are potentially associated with the severity of polyposis in APC1311/+ pigs.
Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/genética , Proteínas de Sinalização Intercelular CCN/genética , Polimorfismo de Nucleotídeo Único , Receptores de Fator Estimulador de Colônias/genética , Polipose Adenomatosa do Colo/patologia , Animais , Proteínas de Sinalização Intercelular CCN/metabolismo , Modelos Animais de Doenças , Mutação , Estabilidade de RNA , Receptores de Fator Estimulador de Colônias/metabolismo , SuínosRESUMO
Familial adenomatous polyposis (FAP) is a hereditary predisposition to formation of colon polyps that can progress to colorectal cancer (CRC). The severity of polyposis varies substantially within families bearing the same germline mutation in the adenomatous polyposis coli (APC) tumour suppressor gene. The progressive step-wise accumulation of genetic events in tumour suppressor genes and oncogenes leads to oncogenic transformation, with driver alterations in the tumour protein p53 (TP53) gene playing a key role in advanced stage CRC. We analysed groups of pigs carrying a truncating mutation in APC (APC1311/+; orthologous to human APC1309/+) to study the influence of TP53 polymorphisms and expression on the frequency of polyp formation and polyp progression in early-stage FAP. Five generations of APC1311/+ pigs were examined by colonoscopy for polyposis severity and development. A total of 19 polymorphisms were found in 5'-flanking, coding, and 3' untranslated regions of TP53. The distribution of TP53 genotypes did not differ between APC1311/+ pigs with low (LP) and high (HP) number of colon polyps. p53 mRNA expression was analysed in distally located normal mucosa samples of wild-type pigs, APC1311/+ LP and HP pigs, and also in distally located polyp samples histologically classified as low-grade (LG-IEN) and high-grade intraepithelial dysplastic (HG-IEN) from APC1311/+ pigs. p53 mRNA expression was found to be significantly elevated in HG-IEN compared to LG-IEN samples (p = 0.012), suggesting a role for p53 in the early precancerous stages of polyp development.
Assuntos
Polipose Adenomatosa do Colo/genética , Pólipos/genética , Proteína Supressora de Tumor p53/genética , Animais , Colo/patologia , Modelos Animais de Doenças , Predisposição Genética para Doença , Genótipo , Mutação de Sentido Incorreto , Polimorfismo de Nucleotídeo Único , SuínosRESUMO
Hereditary spastic paraplegias (HSPs) consist of a heterogeneous group of genetically determined neurodegenerative disorders. Progressive lower extremity weakness and spasticity are the prominent features of HSPs resulting from retrograde axonal degeneration of the corticospinal tracts. Three genetic types, SPG3 (ATL1), SPG4 (SPAST) and SPG31 (REEP1), appear predominantly and may account for up to 50% of autosomal dominant hereditary spastic paraplegias (AD-HSPs). Here, we present the results of genetic testing of the three mentioned SPG genetic types in a group of 216 unrelated Polish patients affected with spastic paraplegia. Molecular evaluation was performed by multiplex ligation-dependent probe amplification (MLPA) and DNA sequencing. Nineteen novel mutations: 13 in SPAST, 4 in ATL1 and 2 in REEP1, were identified among overall 50 different mutations detected in 57 families. Genetic analysis resulted in the identification of molecular defects in 54% of familial and 8.4% of isolated cases. Our research expanded the causative mutations spectrum of the three most common genetic forms of HSPs found in a large cohort of probands originating from the Central Europe.
Assuntos
Adenosina Trifosfatases/genética , Proteínas de Ligação ao GTP/genética , Predisposição Genética para Doença/genética , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Mutação/genética , Paraplegia Espástica Hereditária/genética , Adulto , Análise Mutacional de DNA , Saúde da Família , Feminino , Estudos de Associação Genética , Humanos , Masculino , Polônia , Espastina , Adulto JovemRESUMO
Desmin is a muscle-specific intermediate filament protein which forms a network connecting the sarcomere, T tubules, sarcolemma, nuclear membrane, mitochondria and other organelles. Mutations in the gene coding for desmin (DES) cause skeletal myopathies often combined with cardiomyopathy, or isolated cardiomyopathies. The molecular pathomechanisms of the disease remain ambiguous. Here, we describe and comprehensively characterize two DES mutations found in Polish patients with a clinical diagnosis of desminopathy. The study group comprised 16 individuals representing three families. Two mutations were identified: a novel missense mutation (Q348P) and a small deletion of nine nucleotides (A357_E359del), previously described by us in the Polish population. A common ancestry of all the families bearing the A357_E359del mutation was confirmed. Both mutations were predicted to be pathogenic using a bioinformatics approach, including molecular dynamics simulations which helped to rationalize abnormal behavior at molecular level. To test the impact of the mutations on DES expression and the intracellular distribution of desmin muscle biopsies were investigated. Elevated desmin levels as well as its atypical localization in muscle fibers were observed. Additional staining for M-cadherin, α-actinin, and myosin heavy chains confirmed severe disruption of myofibrill organization. The abnormalities were more prominent in the Q348P muscle, where both small atrophic fibers as well large fibers with centrally localized nuclei were observed. We propose that the mutations affect desmin structure and cause its aberrant folding and subsequent aggregation, triggering disruption of myofibrils organization.