RESUMO
PURPOSE: To evaluate acetylation of histone H3, chromatin remodeling, nuclear size and shape, DNA ploidy, and distribution of nucleolus organizing regions (NORs) in corneal epithelial and stromal cells of diabetic and nondiabetic rats. METHODS: Diabetes was induced by a single intraperitoneal injection of alloxan. All diabetic rats (n = 20) included in the study had 4 weeks of moderate-to-severe hyperglycemia (plasma glucose levels >400 mg/dL). Acetylated histone H3 levels were quantified in corneal tissue using a colorimetric assay. Chromatin remodeling, nuclear sizes (area/perimeter) and shapes (circularity), and DNA ploidies were evaluated from Feulgen-stained tissue sections using video image analysis. Distributions of NORs were studied in tissue sections impregnated with silver ions. Ophthalmic clinical parameters, including corneal sensitivity, were investigated. Twenty nondiabetic rats were used as controls. RESULTS: Acetylation of histone H3 was reduced in the corneas of the diabetic rats. Nuclei in corneal epithelial cells of diabetic rats compacted chromatin, increased in size, modified their shapes, and elevated DNA ploidy. The only nuclear change observed in the corneal stromal cells of diabetic rats was chromatin decompaction. The size of the silver-stained NOR did not differ between the study samples. The corneal sensitivity in diabetic rats was 51.8% lower than that in nondiabetic rats. CONCLUSIONS: The results of this study show that alloxan-induced diabetes altered the histone H3 acetylation pattern and compromised the chromatin supraorganization in corneal tissue/cells. Continued research is needed to understand the clinical and morphofunctional significance of changes in corneal cell nuclei of diabetic individuals.
Assuntos
Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Córnea/metabolismo , Diabetes Mellitus Experimental/metabolismo , Histonas/metabolismo , Acetilação , Aloxano/farmacologia , Animais , Forma do Núcleo Celular/efeitos dos fármacos , Tamanho do Núcleo Celular/efeitos dos fármacos , Diabetes Mellitus Experimental/patologia , Feminino , Ploidias , Ratos , Ratos WistarRESUMO
Metal complexes are still broadly used as the first line of the treatment for different types of tumors nowadays. Carboplatin and oxaliplatin were authorized for clinical use, even though there is little information on the mutagenic profile associated to their usage. This study evaluated the cytostatic effects and the induction of complex genomic alterations after 24-h treatment of CHO-K1 cells to concentrations of 12.5-800 µM of carboplatin and oxaliplatin in the cytokinesis-block micronucleus assay (CBMN-Cyt). The results demonstrated that carboplatin and oxaliplatin significantly increased the frequency of micronuclei (MN), nucleoplasmatic bridges (NPBs), and nuclear buds (NBUDs). On one hand, oxaliplatin induces significantly more chromosomal abnormalities than carboplatin at concentrations of 12.5 and 25 µM. On the other hand, carboplatin, in cells exposed to concentrations of 50 and 100 µM, is more efficient than oxaliplatin in the induction of chromosomal instability events. Both drugs cause significant reduction in the cytokinesis-block proliferation index, demonstrating their cytostatic effects at concentrations 50-800 µM. The results of this study shed more light on the characterization of biological effects associated with the exposure to carboplatin and oxaliplatin.
Assuntos
Antineoplásicos/efeitos adversos , Carboplatina/efeitos adversos , Núcleo Celular/efeitos dos fármacos , Instabilidade Cromossômica/efeitos dos fármacos , Micronúcleos com Defeito Cromossômico/induzido quimicamente , Mutagênicos/efeitos adversos , Compostos Organoplatínicos/efeitos adversos , Animais , Células CHO , Forma do Núcleo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cricetulus , Testes para Micronúcleos , Testes de Mutagenicidade , Concentração Osmolar , OxaliplatinaRESUMO
The deleterious effects of ethanol (EtOH) on the brain have been widely described, but its effects on the neuronal cytoskeleton during differentiation have not yet been firmly established. In this context, our aim was to investigate the direct effect of EtOH on cortical neurons during the period of differentiation. Primary cultures of cortical neurons obtained from 1-day-old rats were exposed to EtOH after 7days of culture, and viability and morphology were analyzed at structural and ultrastructural levels after 24-h EtOH exposure. EtOH caused a significant reduction of 73±7% in the viability of cultured cortical neurons, by preferentially inducing apoptotic cellular death. This effect was accompanied by an increase in caspase 3 and 9 expression. Furthermore, EtOH induced a reduction in total dendrite length and in the number of dendrites per cell. Ultrastructural studies showed that EtOH increased the number of lipidic vacuoles, lysosomes and multilamellar vesicles and induced a dilated endoplasmatic reticulum lumen and a disorganized Golgi apparatus with a ring-shape appearance. Microtubules showed a disorganized distribution. Apposition between pre- and postsynaptic membranes without a defined synaptic cleft and a delay in presynaptic vesicle organization were also observed. Synaptophysin and PSD95 expression, proteins pre- and postsynaptically located, were reduced in EtOH-exposed cultures. Overall, our study shows that EtOH induces neuronal apoptosis and changes in the cytoskeleton and membrane proteins related with the establishment of mature synapses. These direct effects of EtOH on neurons may partially explain its effects on brain development.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Etanol/toxicidade , Neurônios/efeitos dos fármacos , Neurônios/patologia , Sinapses/efeitos dos fármacos , Sinapses/patologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Western Blotting , Forma do Núcleo Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/patologia , Córtex Cerebral/fisiopatologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Imunofluorescência , Marcação In Situ das Extremidades Cortadas , Microscopia Eletrônica , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/metabolismo , Neurogênese/efeitos dos fármacos , Neurogênese/fisiologia , Neurônios/fisiologia , Ratos Wistar , Sinapses/fisiologiaRESUMO
L-asparaginase is an enzyme used as a chemotherapeutic agent, mainly for treating acute lymphoblastic leukemia. In this study, the gene of L-asparaginase from Zymomonas mobilis was cloned in pET vectors, fused to a histidine tag, and had its codons optimized. The L-asparaginase was expressed extracellularly and intracellularly (cytoplasmically) in Escherichia coli in far larger quantities than obtained from the microorganism of origin, and sufficient for initial cytotoxicity tests on leukemic cells. The in silico analysis of the protein from Z. mobilis indicated the presence of a signal peptide in the sequence, as well as high identity to other sequences of L-asparaginases with antileukemic activity. The protein was expressed in a bioreactor with a complex culture medium, yielding 0.13 IU/mL extracellular L-asparaginase and 3.6 IU/mL intracellular L-asparaginase after 4 h of induction with IPTG. The cytotoxicity results suggest that recombinant L-asparaginase from Z. mobilis expressed extracellularly in E.coli has a cytotoxic and cytostatic effect on leukemic cells.
Assuntos
Antineoplásicos/uso terapêutico , Asparaginase/uso terapêutico , Escherichia coli/metabolismo , Leucemia/tratamento farmacológico , Proteínas Recombinantes/uso terapêutico , Zymomonas/enzimologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Asparaginase/genética , Asparaginase/farmacologia , Sequência de Bases , Reatores Biológicos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Forma do Núcleo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Criança , Pré-Escolar , Clonagem Molecular , Simulação por Computador , Feminino , Humanos , Lactente , Leucemia/patologia , Masculino , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologiaRESUMO
Croton membranaceus aqueous root extract (CMARE) is among the widely used phytotherapeutics in Ghana for the management of benign prostatic hyperplasia (BPH) and prostate cancer. However, the mechanism of action of CMARE remains to be elucidated. This study aimed to establish whether apoptosis is involved in the antiproliferative effect of CMARE on human BPH-1 cells. We determined the effect of treatment with 0, 1, 3, and 5 mg/mL CMARE for 24, 48, and 72 h on the viability and morphology of BPH-1 cells using the MMT assay and phase-contrast microscopy, respectively. We examined the apoptosis-inducing effects of CMARE after 48 h at the cellular level using Hoescht 33258 and JC-1 dye staining and flow cytometry analysis. We performed reverse transcription polymerase chain reaction and Western blotting to confirm the apoptotic effects of CMARE at the molecular level. CMARE induced a significant dose-dependent inhibition in the proliferation of BPH-1 cells (P < 0.05) and an alteration in their morphology and a reduction their density. Furthermore, CMARE induced dose-dependent staining of the nuclear chromatin, significant DNA fragmentation with G0/G1 sub-diploid cells (P < 0.01), and loss of the mitochondrial membrane potential in the treated cells compared to the controls after 48 h (P < 0.01). Additionally, while CMARE induced a significant upregulation of the mRNA and protein levels of Bax, those of Bcl2 did not change significantly. Therefore, induction of mitochondria-dependent apoptosis of BPH-1 cells may be a possible mechanism of action of CMARE.
Assuntos
Apoptose/efeitos dos fármacos , Croton/química , Mitocôndrias/metabolismo , Extratos Vegetais/farmacologia , Raízes de Plantas/química , Hiperplasia Prostática/patologia , Bisbenzimidazol , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Forma do Núcleo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citometria de Fluxo , Humanos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
To establish the effect of low (11 mM) and high (55 mM) glucose concentrations (G11, G55) on Jurkat cells exposed to rotenone (ROT, a class 5 mitocan). We demonstrated that ROT induces apoptosis in Jurkat cells cultured in G11 by oxidative stress (OS) mechanism involving the generation of anion superoxide radical (O2(â-), 68%)/hydrogen peroxide (H2O2, 54%), activation of NF-κB (32%), p53 (25%), c-Jun (17%) transcription factors, and caspase-3 (28%), apoptosis-inducing factor (AIF, 36%) nuclei translocation, c-Jun N-terminal kinase (JNK) activation, and loss of mitochondria transmembrane potential (ΔΨm, 62%) leading to nuclei fragmentation (~10% and ~40% stage I-II fragmented nuclei, resp.). ROT induces massive cytoplasmic aggregates of DJ-1 (93%), and upregulation of Parkin compared to untreated cells, but no effect on PINK-1 protein was observed. Cell death marker detection and DJ-1 and Parkin expression were significantly reduced when cells were cultured in G55 plus ROT. Remarkably, metformin sensitized Jurkat cells against ROT in G55. Our results indicate that a high-glucose milieu promotes resistance against ROT/H2O2-induced apoptosis in Jurkat cells. Our data suggest that combined therapy by using mitochondria-targeted damaging compounds and regulation of glucose (e.g., metformin) can efficiently terminate leukemia cells via apoptosis in hyperglycemic conditions.
Assuntos
Glucose/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Modelos Biológicos , Proteínas Oncogênicas/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteínas Quinases/metabolismo , Rotenona/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Caspase 3/metabolismo , Forma do Núcleo Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/metabolismo , Células Jurkat , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Metformina/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteína Desglicase DJ-1 , Transdução de Sinais/efeitos dos fármacos , Superóxidos/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Novel ß-lapachone analogs 2-phenyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione (NQ1), 2-p-tolyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione (NQ3) and 2-methyl-2-phenyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione (NQ7), which have trypanocidal activity, were assayed for cytotoxic effects on murine EL-4 T lymphoma cells. The NQs inhibited the proliferation of EL-4 cells at concentrations above 1µM. Nuclear staining of the EL-4 cells revealed chromatin condensation and a nuclear morphology compatible with the induction of apoptosis. Flow cytometry assays with annexin V-FITC and propidium iodide confirmed the cell death by apoptosis. Using electron paramagnetic resonance (EPR), a semiquinone radical was detected in EL-4 cells treated with NQs. In addition, a decrease in the GSH level in parallel with reactive oxygen species (ROS) production was observed. Preincubation with n-acetyl-l-cysteine (NAC) was able to reverse the inhibitory effects of the NQs on cell proliferation, indicating that ROS generation is involved in NQ-induced apoptosis. In addition, the NQs induced a decrease in the mitochondrial membrane potential and increased the proteolytic activation of caspases 9 and 3 and the cleavage of Poly (ADP-Ribose) Polymerase (PARP). In conclusion, these results indicate that redox cycling is induced by the NQs in the EL-4 cell line, with the generation of ROS and other free radicals that could inhibit cellular proliferation as a result of the induction of the intrinsic apoptosis pathway.