RESUMO
During tumorigenesis, the mechanical properties of cancer cells change markedly, with decreased stiffness often accompanying a more invasive phenotype. Less is known about the changes in mechanical parameters at intermediate stages in the process of malignant transformation. We have recently developed a pre-tumoral cell model by stably transducing the immortalized but non-tumorigenic human keratinocyte cell line HaCaT with the E5, E6 and E7 oncogenes from HPV-18, one of the leading causes of cervical cancer and other types of cancer worldwide. We have used atomic force microscopy (AFM) to measure cell stiffness and to obtain mechanical maps of parental HaCaT and HaCaT E5/E6/E7-18 cell lines. We observed a significant decrease in Young's modulus in HaCaT E5/E6/E7-18 cells measured by nanoindentation in the central region, as well as decreased cell rigidity in regions of cell-cell contact measured by Peakforce Quantitative Nanomechanical Mapping (PF-QNM). As a morphological correlate, HaCaT E5/E6/E7-18 cells displayed a significantly rounder cell shape than parental HaCaT cells. Our results therefore show that decreased stiffness with concomitant perturbations in cell shape are early mechanical and morphological changes during the process of malignant transformation.
Assuntos
Proteínas Oncogênicas Virais , Feminino , Humanos , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Papillomavirus Humano 18/genética , Proteínas E7 de Papillomavirus/metabolismo , Proteínas Repressoras/metabolismo , Oncogenes , Transformação Celular Neoplásica/genética , Queratinócitos/metabolismoRESUMO
Individuals with diabetes mellitus (DM) tend to manifest anxiety and depression, which could be related to changes in the expression of calcium/calmodulin-dependent protein kinase IV (CaMKIV), transcription factor cyclic AMP-responsive element binding protein (CREB), phosphorylated CREB (pCREB) and brain-derived neurotrophic factor (BDNF) in different brain regions. The objective of this study was to determine whether mice with type 1 diabetes (T1DM) induced with streptozotocin show a profile of anxious-type behaviors and alterations in the expression/activity of CaMKIV, CREB, pCREB and BDNF in different regions of the brain (prefrontal cortex, amygdala, hippocampus and hypothalamus) in comparison to non-diabetic mice (NDB). Mice with 3 months of chronic DM showed an anxious-like behavioral profile in two anxiety tests (Open Field and Elevated Plus Maze), when compared to NDB. There were significant differences in the expression of cell signaling proteins: diabetic mice had a lower expression of CaMKIV in the hippocampus, a greater expression of CREB in the amygdala and hypothalamus, as well as a lower pCREB/CREB in hypothalamus than NDB mice (P < 0.05). This is the first study evaluating the expression of CaMKIV in the brain of animals with DM, who presented lower expression of this protein in the hippocampus. In addition, it is the first time that CREB was evaluated in amygdala and hypothalamus of animals with DM, who presented a higher expression. Further research is necessary to determine the possible link between expression of CaMKIV and CREB, and the behavioral profile of anxiety in diabetic animals.
Assuntos
Fator Neurotrófico Derivado do Encéfalo , Diabetes Mellitus , Tonsila do Cerebelo , Animais , Ansiedade/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Cálcio/metabolismo , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/metabolismo , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Diabetes Mellitus/metabolismo , Hipocampo/metabolismo , Hipotálamo/metabolismo , Camundongos , Córtex Pré-Frontal/metabolismo , Transdução de Sinais , Estreptozocina/metabolismoRESUMO
H9c2 cardiac cells were incubated under the control condition and at different hyperglycemic and hyperlipidemic media, and the following parameters were determined and quantified: a) cell death, b) type of cell death, and c) changes in cell length, width and height. Of all the proven media, the one that showed the greatest differences compared to the control was the medium glucose (G) 33 mM + 500 µM palmitic acid. This condition was called the hyperglycemic and hyperlipidemic condition (HHC). Incubation of H9c2 cells in HHC promoted 5.2 times greater total cell death when compared to the control. Of the total death ofthe HHC cells, 38.6% was late apoptotic and 8.3% early apoptotic. HHC also changes cell morphology. The reordering of the actin cytoskeleton and cell stiffness was also studied in control and HHC cells. The actin cytoskeleton was quantified and the number and distance of actin bundles were not the same in the control as under HHC. Young's modulus images show a map of cell stiffness. Cells incubated in HHC with the reordered actin cytoskeleton were stiffer than those incubated in control. The region of greatest stiffness was the peripheral zone of HHC cells (where the number of actin bundles was higher and the distance between them smaller). Our results suggest a correlation between the reordering of the actin cytoskeleton and cell stiffness. Thus, our study showed that HHC can promote morphophysiological changes in rat cardiac cells confirming that gluco-and lipotoxicity may play a central role in the development of diabetic cardiomyopathy.
RESUMO
BACKGROUND: Multiple Myeloma (MM) is a malignant hematologic disorder and the second most common blood cancer. Interleukin-6 (IL-6) has been identified as a crucial factor for the proliferation and survival of MM cells and the overexpression of IL-6 receptor is being studied as a molecular target for therapeutic and diagnostic use in myelomas and other comorbidities. Tocilizumab is a humanized monoclonal antibody that binds IL-6R. OBJECTIVE: We aim to label and evaluate Fab(Tocilizumab) with 99mTechnetium or Cy7 as potential MM imaging agents. METHODS: IL-6R distribution was analyzed by Laser Confocal Microscopy (LCM) in MM cell lines. Fab(Tocilizumab) was produced by the digestion of Tocilizumab with papain for 24h at 37°C, derivatized with NHS-HYNIC-Tfa and radiolabeled with 99mTc. Radiochemical stability and in vitro cell assays were evaluated. Biodistribution and SPECT/CT were performed. Also, Fab(Tocilizumab) was labeled with Cy7 for in vivo fluorescence imaging up to 72h. RESULTS: LCM analysis demonstrates IL-6R distribution on MM cell lines. Incubation with papain resulted in complete digestion of Tocilizumab and exhibited a good purity and homogeneity. Radiolabeling with 99mTc via NHS-HYNIC-Tfa was found to be fast, easy, reproducible and stable, revealing high radiochemical purity and without interfering with IL-6R recognition. Biodistribution and SPECT/CT studies showed a quick blood clearance and significant kidney and MM engrafted tumor uptake. Cy7-Fab(Tocilizumab) fluorescent imaging allowed MM1S tumor identification up to 72h p.i. CONCLUSION: These new molecular imaging agents could potentially be used in the clinical setting for staging and follow-up of MM through radioactive whole-body IL-6R expression visualization in vivo. The fluorescent version could be used for tissue sample evaluation and to guide surgical excision, if necessary.
Assuntos
Anticorpos Monoclonais Humanizados/química , Carbocianinas/química , Imagem Molecular , Mieloma Múltiplo/diagnóstico por imagem , Compostos de Organotecnécio/química , Compostos Radiofarmacêuticos/química , Humanos , Receptores de Interleucina-6/análiseRESUMO
Eugenia involucrata DC. (Myrtaceae) is a native tree species from Brazil that has been scarcely studied. We investigated the phenolic composition, the antioxidant capacity and the antitumoral activity of ethanolic extracts from fruits (FE) and seeds (SE) of E. involucrata. Six anthocyanins were identified by UPLC-PDA/MS/MS in FE, being four derived from cyanidin, and the other ones derived from delphinidin and pelargonidin. Using HPLC-PDA, FE presented a larger number of phenolic compounds (epicatechin, catechin, rutin, ellagic acid, myricetin and quercetin) than SE, which did not show myricetin and quercetin. However, SE showed higher total phenolic content and generally stronger in vitro antioxidant capacity than FE, except that only FE exhibited superoxide radical scavenging activity, which may be attributed to the anthocyanins present in fruits. Additionally, only SE exhibited antitumoral activity in a pancreatic cancer cell line (PANC-1). The antitumoral mechanisms involved imbalance of antioxidant status, alteration of mitochondrial membrane potential, cytoskeleton disassembly and induction of cell death by apoptosis and necrosis. Compared to the standard antitumoral drug gemcitabine, SE exhibited higher antitumoral efficacy and selectivity index. The highest concentration of total phenolics and of specific phenolic compounds bearing antitumoral properties may be related to the antitumoral activity of SE. Our results corroborate previous data of E. involucrata as an important source of bioactive compounds and provide, for the first time, evidences of in vitro antitumoral potential of its seeds on pancreatic cancer cell line.
Assuntos
Antioxidantes , Eugenia , Antioxidantes/farmacologia , Brasil , Frutas , Extratos Vegetais/farmacologia , Sementes , Espectrometria de Massas em TandemRESUMO
Studies have shown the cytoskeleton disorganization produced by diabetes and quantified F-actin fluorescence in the striated muscles of diabetic animals. However, at present, there are no studies that have quantified F-actin spatial organization (F-actin-SO). Through our research, we analyzed the effect of diabetes on F-actin-SO in the cardiac and skeletal muscles of a mouse model. The muscle samples were labeled with phalloidin-rhodamine and analyzed with confocal microscopy. The analysis was done in two dimensions using four approaches: quantitation of (a) phalloidin-occupied areas; (b) number of F-actin-unoccupied areas per muscular fiber; (c) F-actin filament discontinuity; and (d) costamere periodicity. Our results showed that both the cardiac and skeletal muscles of the control mice had more phalloidin-occupied areas than the diabetic mice. The skeletal muscles had a significantly higher number of F-actin-unoccupied areas per muscular fiber and more F-actin discontinuities. Additionally, using western blot analyses, we showed that those differences were not due to α-actin protein expression. Finally, we considered the importance of these findings in dysfunctional contraction, disassembly in cell-cell communication, conduction of muscle impulse, and changes in cell nanomechanics. Our results quantitatively demonstrated that diabetes severely affects F-actin-SO in striated muscles.
Assuntos
Actinas/metabolismo , Citoesqueleto/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Músculo Estriado/metabolismo , Animais , Masculino , Camundongos , RatosRESUMO
PURPOSE: To study the rhenium-188 labeling of polyamidoamine (PAMAM) generation 4 (G4) dendrimer and its evaluation on biodistribution and chromosomal aberrations in melanoma cells induced by ionizing radiation as potential treatment agent. MATERIALS AND METHODS: Dendrimers were first conjugated with Suc-HYNIC (succinimidyl 6-hydrazinopyridine-3-carboxylic acid hydrochloride). Dendrimer-HYNIC was then incubated with 188ReO4-. Biodistribution was performed administrating 188Re-dendrimer to normal (NM) or melanoma-bearing mice (MBM). Chromosome aberration test was conducted in order to measure treatment capacity of 188Re-dendrimer in melanoma cells. RESULTS: Radiolabeling yield of dendrimer was approx. 70%. Biodistribution studies in NM showed blood clearance with hepatic and renal depuration. MBM showed a similar pattern of biodistribution with tumor uptake of 6% of injected dose. Aberrant metaphases quantified in control cells were 7%, increasing to 29.5% in cells treated with 15µCi (0.555 MBq) of 188Re-dendrimer for 24 h. CONCLUSIONS: 188Re-dendrimer can produce double-stranded breaks in DNA induced by ionizing radiation in melanoma cells in vitro.
Assuntos
Aberrações Cromossômicas/efeitos da radiação , Dendrímeros/química , Melanoma Experimental/radioterapia , Radioisótopos/toxicidade , Rênio/toxicidade , Animais , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Marcação por Isótopo , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Radioisótopos/farmacocinética , Rênio/farmacocinética , Distribuição TecidualRESUMO
Nuclear lamins support the nuclear envelope and provide anchorage sites for chromatin. They are involved in DNA synthesis, transcription, and replication. It has previously been reported that the lack of Lamin A/C expression in lymphoma and leukaemia is due to CpG island promoter hypermethylation. Here, we provide evidence that Lamin A/C is silenced via this mechanism in a subset of neuroblastoma cells. Moreover, Lamin A/C expression can be restored with a demethylating agent. Importantly, Lamin A/C reintroduction reduced cell growth kinetics and impaired migration, invasion, and anchorage-independent cell growth. Cytoskeletal restructuring was also induced. In addition, the introduction of lamin Δ50, known as Progerin, caused senescence in these neuroblastoma cells. These cells were stiffer and developed a cytoskeletal structure that differed from that observed upon Lamin A/C introduction. Of relevance, short hairpin RNA Lamin A/C depletion in unmethylated neuroblastoma cells enhanced the aforementioned tumour properties. A cytoskeletal structure similar to that observed in methylated cells was induced. Furthermore, atomic force microscopy revealed that Lamin A/C knockdown decreased cellular stiffness in the lamellar region. Finally, the bioinformatic analysis of a set of methylation arrays of neuroblastoma primary tumours showed that a group of patients (around 3%) gives a methylation signal in some of the CpG sites located within the Lamin A/C promoter region analysed by bisulphite sequencing PCR. These findings highlight the importance of Lamin A/C epigenetic inactivation for a subset of neuroblastomas, leading to enhanced tumour properties and cytoskeletal changes. Additionally, these findings may have treatment implications because tumour cells lacking Lamin A/C exhibit more aggressive behaviour.
Assuntos
Neoplasias Encefálicas/genética , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Lamina Tipo A/genética , Neuroblastoma/genética , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Sequência de Bases , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Ilhas de CpG , Humanos , Lamina Tipo A/antagonistas & inibidores , Lamina Tipo A/metabolismo , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Neuroglia/metabolismo , Neuroglia/patologia , Neurônios/metabolismo , Neurônios/patologia , Cultura Primária de Células , Regiões Promotoras Genéticas , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Breast cancer is the second leading cause of cancer death worldwide. Nanotechnology approaches can overcome the side effects of chemotherapy as well as improve the efficacy of drugs. Dendrimers are nanometric size polymers which are suitable as drug delivery systems. To the best of our knowledge, studies on the application of PAMAM G4.5 (polyamidoamine half generation 4) dendrimers as potential drug delivery systems in breast cancer have not been reported. In this work we developed a PAMAM G4.5 dendrimer containing FITC (fluorescein isothiocyanate) dye to study their uptake by murine breast cancer cells and BALB/c mice breast tumors. RESULTS: We performed a reaction between FITC and PAMAM G4.5 dendrimers which were previously derivatized with piperazine (linker molecule), characterized them by (1)H NMR (proton nuclear magnetic resonance) spectroscopy and MALDI-TOF (matrix-assisted laser desorption/ionization- time-of-flight) mass spectrometry. The experimental data indicated that 2 FITC molecules could be bound covalently at the PAMAM G4.5 dendrimer surface, with 17 FITC molecules probably occluded in PAMAM dendrimers cavity. PAMAM-FITC dendrimer (PAMAM G4.5-piperazinyl-FITC dendrimer) size distribution was evaluated by DLS (dynamic light scattering) and TEM (transmission electron microscopy). The nanoparticle hydrodynamic size was 96.3 ± 1.4 nm with a PdI (polydispersion index) of 0.0296 ± 0.0171, and the size distribution measured by TEM was 44.2 ± 9.2 nm. PAMAM-FITC dendrimers were neither cytotoxic in 4T1 cells nor hemolytic up to 24 h of incubation. In addition, they were uptaken in vitro by 4T1 cells and in vivo by BALB/c mice breast tumors. PAMAM G4.5-piperazinyl-FITC dendrimer intracellular distribution was observed through histologic analysis of the tumor by laser confocal microscopy. CONCLUSION: These results indicate that PAMAM G4.5 dendrimers enter tumor tissue cells, being good candidates to be used as antitumor drug delivery systems for breast cancer treatment and diagnosis.
Assuntos
Antineoplásicos/administração & dosagem , Dendrímeros/metabolismo , Portadores de Fármacos/metabolismo , Fluoresceína-5-Isotiocianato/administração & dosagem , Corantes Fluorescentes/administração & dosagem , Nylons/metabolismo , Animais , Antineoplásicos/farmacocinética , Mama/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Fluoresceína-5-Isotiocianato/farmacocinética , Corantes Fluorescentes/farmacocinética , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Superparamagnetic iron oxide nanoparticles (SPIONS) were synthesized by thermal decomposition of an organometallic precursor at high temperature and coated with a bi-layer composed of oleic acid and methoxy-polyethylene glycol-phospholipid. The formulations were named SPION-PEG350 and SPION-PEG2000. Transmission electron microscopy, X-ray diffraction and magnetic measurements show that the SPIONs are near-spherical, well-crystalline, and have high saturation magnetization and susceptibility. FTIR spectroscopy identifies the presence of oleic acid and of the conjugates mPEG for each sample. In vitro biocompatibility of SPIONS was investigated using three cell lines; up to 100µg/ml SPION-PEG350 showed non-toxicity, while SPION-PEG2000 showed no signal of toxicity even up to 200µg/ml. The uptake of SPIONS was detected using magnetization measurement, confocal and atomic force microscopy. SPION-PEG2000 presented the highest internalization capacity, which should be correlated with the mPEG chain size. The in vivo results suggested that SPION-PEG2000 administration in mice triggered liver and kidney injury. FROM THE CLINICAL EDITOR: The potential use of superparamagnetic iron oxide nanoparticles (SPIONS) in the clinical setting have been studied by many researchers. The authors synthesized two types of SPIONS here and investigated the physical properties and biological compatibility. The findings should provide more data on the design of SPIONS for clinical application in the future.