RESUMO
Stromal interaction molecule 1 (STIM1) and the ORAI1 calcium channel mediate store-operated calcium entry (SOCE) and regulate a multitude of cellular functions. The identity and function of these proteins in thyroid cancer remain elusive. We show that STIM1 and ORAI1 expression is elevated in thyroid cancer cell lines, compared to primary thyroid cells. Knock-down of STIM1 or ORAI1 attenuated SOCE, reduced invasion, and the expression of promigratory sphingosine 1-phosphate and vascular endothelial growth factor-2 receptors in thyroid cancer ML-1 cells. Cell proliferation was attenuated in these knock-down cells due to increased G1 phase of the cell cycle and enhanced expression of cyclin-dependent kinase inhibitory proteins p21 and p27. STIM1 protein was upregulated in thyroid cancer tissue, compared to normal tissue. Downregulation of STIM1 restored expression of thyroid stimulating hormone receptor, thyroid specific proteins and increased iodine uptake. STIM1 knockdown ML-1 cells were more susceptible to chemotherapeutic drugs, and significantly reduced tumor growth in Zebrafish. Furthermore, STIM1-siRNA-loaded mesoporous polydopamine nanoparticles attenuated invasion and proliferation of ML-1 cells. Taken together, our data suggest that STIM1 is a potential diagnostic and therapeutic target for treatment of thyroid cancer.
Assuntos
Proliferação de Células/genética , Proteínas de Neoplasias/genética , Molécula 1 de Interação Estromal/genética , Células Epiteliais da Tireoide/patologia , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Canais de Cálcio/genética , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Fase G1/efeitos dos fármacos , Fase G1/genética , Humanos , Indóis/administração & dosagem , Masculino , Pessoa de Meia-Idade , Nanopartículas/administração & dosagem , Proteína ORAI1/genética , Polímeros/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Células Epiteliais da Tireoide/efeitos dos fármacos , Glândula Tireoide/efeitos dos fármacos , Neoplasias da Glândula Tireoide/tratamento farmacológico , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Adulto Jovem , Peixe-ZebraRESUMO
The major barriers for intracellular DNA transportation by cationic polymers are their toxicity, poor endosomal escape and inefficient nuclear uptake. Therefore, we designed novel modular peptide-based carriers modified with SV40 nuclear localization signal (NLS). Core peptide consists of arginine, histidine and cysteine residues for DNA condensation, endosomal escape promotion and interpeptide cross-linking, respectively. We investigated three polyplexes with different NLS content (10â¯mol%, 50â¯mol% and 90â¯mol% of SV40 NLS) as vectors for intranuclear DNA delivery. All carriers tested were able to condense DNA, to protect it from DNAase I and were not toxic to the cells. We observed that cell cycle arrest by hydroxyurea did not affect transfection efficacy of NLS-modified carriers which we confirmed using quantitative confocal microscopy analysis. Overall, peptide carrier modified with 90â¯mol% of SV40 NLS provided efficient transfection and nuclear uptake in non-dividing cells. Thus, incorporation of NLS into arginine-rich cross-linking peptides is an adequate approach to the development of efficient intranuclear gene delivery vehicles.
Assuntos
Arginina/metabolismo , DNA/metabolismo , Sinais de Localização Nuclear/metabolismo , Peptídeos/metabolismo , Vírus 40 dos Símios/metabolismo , DNA/química , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Células HeLa , Humanos , Hidroxiureia/toxicidade , Microscopia Confocal , Sinais de Localização Nuclear/química , Peptídeos/química , Peptídeos/toxicidade , TransfecçãoRESUMO
The 9p21.3 genomic locus is a hot spot for disease-associated single-nucleotide polymorphisms (SNPs), and its strongest associations are with coronary artery disease (CAD). The disease-associated SNPs are located within the sequence of a long noncoding RNA ANRIL, which potentially contributes to atherogenesis by regulating vascular cell stress and proliferation, but also affects pancreatic ß-cell proliferation. Altered expression of a neighboring gene, CDKN2B, has been also recognized to correlate with obesity and hepatic steatosis in people carrying the risk SNPs. In the present study, we investigated the impact of 9p21.3 on obesity accompanied by hyperlipidemia in mice carrying a deletion of the murine ortholog for the 9p21.3 (Chr4Δ70/Δ70) risk locus in hyperlipidemic Ldlr-/-ApoB100/100 background. The Chr4Δ70/Δ70 mice showed decreased mRNA expression of insulin receptors in white adipose tissue already at a young age, which developed into insulin resistance and obesity by aging. In addition, the Sirt1-Ppargc1a-Ucp2 pathway was downregulated together with the expression of Cdkn2b, specifically in the white adipose tissue in Chr4Δ70/Δ70 mice. These results suggest that the 9p21.3 locus, ANRIL lncRNA, and their murine orthologues may regulate the key energy metabolism pathways in a white adipose tissue-specific manner in the presence of hypercholesterolemia, thus contributing to the pathogenesis of metabolic syndrome.
Assuntos
Hipercolesterolemia , Resistência à Insulina , Obesidade , Animais , Obesidade/genética , Obesidade/metabolismo , Resistência à Insulina/genética , Hipercolesterolemia/genética , Hipercolesterolemia/metabolismo , Hipercolesterolemia/complicações , Camundongos , Humanos , Cromossomos Humanos Par 9/genética , Masculino , Deleção de Genes , Loci Gênicos , Camundongos Endogâmicos C57BL , Tecido Adiposo Branco/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
Due to its modular and flexible design options, mesoporous silica provides ample opportunities when developing new strategies for combinatory antibacterial treatments. In this study, antibacterial ceria (CeO2) nanoparticles (NP) were used as core material, and were further coated with a mesoporous silica shell (mSiO2) to obtain a core@shell structured nanocomposite (CeO2@mSiO2). The porous silica shell was utilized as drug reservoir, whereby CeO2@mSiO2 was loaded with the antimicrobial agent capsaicin (CeO2@mSiO2/Cap). CeO2@mSiO2/Cap was further surface-coated with the natural antimicrobial polymer chitosan by employing physical adsorption. The obtained nanocomposite, CeO2@mSiO2/Cap@Chit, denoted NAB, which stands for "nanoantibiotic", provided a combinatory antibacterial mode of action. The antibacterial effect of NAB on the Gram-negative bacteria Escherichia coli (E.coli) was proven to be significant in vitro. In addition, in vivo evaluations revealed NAB to inhibit the bacterial growth in the intestine of bacteria-fed Drosophila melanogaster larvae, and decreased the required dose of capsaicin needed to eliminate bacteria. As our constructed CeO2@mSiO2 did not show toxicity to mammalian cells, it holds promise for the development of next-generation nanoantibiotics of non-toxic nature with flexible design options.
Assuntos
Nanopartículas , Dióxido de Silício , Animais , Antibacterianos/farmacologia , Capsaicina , Drosophila melanogaster , Mamíferos , Nanopartículas/uso terapêutico , Dióxido de Silício/farmacologiaRESUMO
Gene therapy using siRNA molecules is nowadays considered as a promising approach. For successful therapy, development of a stable and reliable vector for siRNA is crucial. Non-viral and non-organic vectors like mesoporous silica nanoparticles (MSN) are associated with lack of most viral vector drawbacks, such as toxicity, immunogenicity, but also generally a low nucleic acid carrying capacity. To overcome this hurdle, we here modified the pore walls of MSNs with surface-hyperbranching polymerized poly(ethyleneimine) (hbPEI), which provides an abundance of amino-groups for loading of a larger amount of siRNA molecules via electrostatic adsorption. After loading, the particles were covered with a second layer of pre-polymerized PEI to provide better protection of siRNA inside the pores, more effective cellular uptake and endosomal escape. To test the transfection efficiency of PEI covered siRNA/MSNs, MDA-MB 231 breast cancer cells stably expressing GFP were used. We demonstrate that PEI-coated siRNA/MSN complexes provide more effective delivery of siRNAs compared to unmodified MSNs. Thus, it can be concluded that appropriately surface-modified MSNs can be considered as prospective vectors for therapeutic siRNA delivery.