RESUMEN
Using biocompatible polymer nanofibrous conduits with a controlled drug delivery have attracted much attention for peripheral nerve regeneration. This work was aimed at preparing electrospun poly (l-lactide-co-D, l-lactide) (PLDLLA) containing multi-walled carbon nanotubes (MWCNTs) and 4-aminopyridine (4-AP)-loaded molecularly imprinted nanoparticles (MIP4-AP) as well as evaluating their performance in in vitro and in vivo assessments. After synthesis of MIP4-AP based on poly (methacrylic acid) with imprinting factor of 1.78, it was incorporated into the PLDLLA/MWCNTs nanofibers to optimize. By adjusting the process variables, the average diameter and electrical conductivity of the nanofibrous sample were 92 nm and 2870 × 10-7 S cm-1, respectively. Afterward, 4-AP release of the optimum sample showed the presence of MIP4-AP leading to initial burst release decrease and plateau level postpone up to 96 h. Moreover, the culture results of PC12 as neuroblastoma cell line on optimal PLDLLA/MWCNTs/MIP4-AP nanofibrous sample revealed the highest cell proliferation without cytotoxicity compared to neat nanofibers. Eventually, the animal model experiment exhibited that the conductive conduit based on the optimum sample was able to repair the rat's sciatic nerve after four weeks in accordance with sciatic function index and histological studies.
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4-Aminopiridina/química , Metacrilatos/química , Impresión Molecular , Nanofibras/química , Regeneración Nerviosa/fisiología , Nervios Periféricos/fisiología , Poliésteres/química , Ingeniería de Tejidos , Adsorción , Animales , Muerte Celular , Proliferación Celular , Supervivencia Celular , Conductividad Eléctrica , Nanofibras/ultraestructura , Nanopartículas/química , Nanopartículas/ultraestructura , Nanotubos de Carbono/química , Nanotubos de Carbono/ultraestructura , Células PC12 , Ratas , Ratas WistarRESUMEN
Aim: Several types of nanocarriers, most of which show significant cytotoxicity, have been developed to overcome the problem of gene-delivery barriers. Biocompatibility, low toxicity and water solubility of carbon nanodots (CNDs) are major advantages that recommend them as delivery systems. Materials & methods: We present a simple method to produce positively charged CNDs. Ethanolamine, ethylenediamine and hydrogen peroxide were utilized to synthesize these CNDs. Results & conclusion: Our results indicated that delivery of the CND-siGFP complex led to significant switching-off of the fluorescence of the GFP-expressing A549 cell. Next, the A549 cells were transfected with siRNA against BiP, which is a pivotal protein in the chemotherapy resistance of cancer cells. The expression levels of BiP decreased remarkably.
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CarbonoRESUMEN
Cisplatin treatment confers the relative resistance to MCF-7 cells as compared to other breast cancer cell lines. One principal reason is that chemotherapeutic agents induce autophagy in these cells to inhibit apoptosis. Binding immunoglobulin protein (BiP), a master regulator of unfolded protein response (UPR) and 14-3-3ζ are two critical proteins upregulated in breast cancer rendering resistance to anticancer drugs. They also play pivotal roles in autophagy with crosstalk with the apoptotic pathways of UPR through certain regulators. Thus, BiP and 14-3-3ζ were selected as the candidate targets to enhance cell death and apoptosis. First, cisplatin resistance was induced and determined by MTT assay and qPCR in MCF-7 cells. Then, the apoptosis axis of UPR was activated by knocking down either BiP or 14-3-3ζ and overactivated by co-knockdown of BiP and 14-3-3ζ. Apoptosis assays were performed using flow cytometry, TUNEL assays utilized confocal microscopy followed by western blot analysis and caspase-3 and JNK activities were investigated to assess the outcomes. Finally, an autophagy assay followed by western blotting was performed to study the effects of co-knockdown genes on cell autophagy in the presence and absence of cisplatin. The present data indicated the enhancement of cisplatin sensitivity in MCF-7 cells co-knocked down in BiP and 14-3-3ζ compared with either gene knockdown. Upregulation of JNK and cleaved-PARP1 protein levels as well as caspase-3 and JNK overactivation confirmed the results. A marked attenuation of autophagy and Beclin1 as well as ATG5 downregulation were detected in co-knockdown cells compared to knockdown with either BiP or 14-3-3ζ. Cisplatin sensitization of MCF-7 cells through double-knockdown of BiP and 14-3-3ζ highlights the potential of targeting UPR and autophagy factors to increase the effect of chemotherapy.
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Proteínas 14-3-3/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Cisplatino/farmacología , Resistencia a Antineoplásicos/genética , Proteínas de Choque Térmico/metabolismo , Proteínas 14-3-3/genética , Apoptosis/efectos de los fármacos , Apoptosis/genética , Autofagia/efectos de los fármacos , Autofagia/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Cisplatino/uso terapéutico , Chaperón BiP del Retículo Endoplásmico , Femenino , Técnicas de Silenciamiento del Gen , Proteínas de Choque Térmico/genética , Humanos , Células MCF-7 , Regulación hacia ArribaRESUMEN
BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cancer worldwide. They are typically characterized by a high incidence of local recurrence, which is the most common cause of death in HNSCC patients. TP53 is the most frequently mutated gene in HNSCC and patients carrying TP53 mutations are associated with a higher probability to develop local recurrence. MiRNAs, which are among the mediators of the oncogenic activity of mt-p53 protein, emerge as an appealing tool for screening, diagnosis and prognosis of cancer. We previously identified a signature of 12 miRNAs whose aberrant expression associated with TP53 mutations and was prognostic for HNSCC. Among them miR-96-5p emerges as an oncogenic miRNAs with prognostic significance in HNSCC. METHODS: To evaluate the oncogenic role of miR-96-5p in a tumoral context, we performed colony formation, cell migration and cell viability assays in two HNSCC cell lines transfected for miR-96-5p mimic or inhibitor and treated with or without radio/chemo-therapy. In addition, to identify genes positively and negatively correlated to miR-96-5p expression in HNSCC, we analyzed the correlation between gene expression and miR-96-5p level in the subset of TCGA HNSCC tumors carrying missense TP53 mutations by Spearman and Pearson correlation. To finally identify targets of miR-96-5p, we used in silico analysis and the luciferase reporter assay to confirm PTEN as direct target. RESULTS: Our data showed that overexpression of miR-96-5p led to increased cell migration and radio-resistance, chemotherapy resistance in HNSCC cells. In agreement with these results, among the most statistically significant pathways in which miR-96-5p is involved, are focal Adhesion, extracellular matrix organization and PI3K-Akt-mTOR-signaling pathway. As a direct target of miR-96-5p, we identified PTEN, the main negative regulator of PI3K-Akt signalling pathway activation. CONCLUSIONS: These results highlight a new mechanism of chemo/radio-resistance insurgence in HNSCC cells and support the possibility that miR-96-5p expression could be used as a novel promising biomarker to predict radiotherapy response and local recurrence development in HNSCC patients. In addition, the identification of pathways in which miR-96-5p is involved could contribute to develop new therapeutic strategies to overcome radio-resistance.
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Resistencia a Antineoplásicos , Neoplasias de Cabeza y Cuello/genética , MicroARNs/genética , Fosfohidrolasa PTEN/genética , Tolerancia a Radiación , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Regiones no Traducidas 3' , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Pronóstico , Transducción de Señal , Análisis de SupervivenciaRESUMEN
Tamoxifen (Nolvadex) is one of the most widely used and effective therapeutic agent for breast cancer. It benefits nearly 75% of patients with estrogen receptor (ER)-positive breast cancer that receive this drug. Its effectiveness is mainly attributed to its capacity to function as an ER antagonist, blocking estrogen binding sites on the receptor, and inhibiting the proliferative action of the receptor-hormone complex. Although, tamoxifen can induce apoptosis in breast cancer cells via upregulation of pro-apoptotic factors, it can also promote uterine hyperplasia in some women. Thus, tamoxifen as a multi-functional drug could have different effects on cells based on the utilization of effective concentrations or availability of specific co-factors. Evidence that tamoxifen functions as a GPR30 (G-Protein Coupled Receptor 30) agonist activating adenylyl cyclase and EGFR (Epidermal Growth Factor Receptor) intracellular signaling networks, provides yet another means of explaining the multi-functionality of tamoxifen. Here ordinary differential equation (ODE) modeling, RNA sequencing and real time qPCR analysis were utilized to establish the necessary data for gene network mapping of tamoxifen-stimulated MCF-7 cells, which express the endogenous ER and GPR30. The gene set enrichment analysis and pathway analysis approaches were used to categorize transcriptionally upregulated genes in biological processes. Of the 2,713 genes that were significantly upregulated following a 48 h incubation with 250 µM tamoxifen, most were categorized as either growth-related or pro-apoptotic intermediates that fit into the Tp53 and/or MAPK signaling pathways. Collectively, our results display that the effects of tamoxifen on the breast cancer MCF-7 cell line are mediated by the activation of important signaling pathways including Tp53 and MAPKs to induce apoptosis.
RESUMEN
Zinc oxide nanoparticles are very toxic, but their agglomeration reduces their lethal cytotoxic effects. Here we tested the hypothesis that conjugation of ZnO nanoparticles via Meso-Tetra (4-Carboxyphenyl) Porphyrin (MTCP) could provide electrostatic or steric stabilization of ZnO nanoparticles and increase their cytotoxic effects. The cytotoxicity and cell death induction were assessed using two human breast adenocarcinoma cell lines (MCF-7 and MDA-MB-468). The MTT results indicated that the toxicity of ZnO nanoparticles was significantly increased upon MTCP conjugation. Annexin/PI and real time RT-PCR results demonstrated that the ZnO-MTCP nanoparticles induced cell death via different non-canonical pathways that are under ca2+ control. Calcium signaling could regulate lysosomal dependent apoptosis and death autophagy, and killing of the two selected types of breast cancer cells.
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Nanopartículas del Metal , Adenocarcinoma , Apoptosis , Autofagia , Neoplasias de la Mama , Línea Celular , Supervivencia Celular , Humanos , Óxido de ZincRESUMEN
Graphene quantum dots (GQDs) are new efficient nanomaterials used in therapeutic applications. In this study, blue fluorescent nitrogen-doped GQDs (N-GQDs) were synthesized by a hydrothermal method via pyrolisis of citric acid as the carbon source and urea as the nitrogen source. The existence of doped nitrogen in GQDs was confirmed by FTIR characterization. Here, for the first time, the N-GQDs were loaded with the anticancer drug, methotrexate (MTX), to prepare MTX-(N-GQDs) as an efficient drug delivery system. The establishment of the strong π-π stacking interaction between MTX and N-GQDs was confirmed by FTIR and UV-vis spectroscopies indicating successful loading of MTX to N-GQDs. The in-vitro cytotoxicity of MTX-(N-GQDs) on human breast cancer cells investigated through MTT assay suggested that the drug-free N-GQDs nanocarriers are highly biocompatible, whereas the MTX-loaded ones are more cytotoxic than the free MTX.
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Puntos Cuánticos , Antineoplásicos , Sistemas de Liberación de Medicamentos , Grafito , Humanos , Metotrexato , Nanoestructuras , NitrógenoRESUMEN
Polymer architecture can influence biodistribution and the mode of presentation of bioactive agents to cells. Herein delivery, loading efficiency, and mode of cellular entry of polymer conjugates of the photosensitizer Meso-Tetra (4-Carboxyphenyl) Porphyrine (MTCP) are examined when attached to hyperbranched amine terminated poly(amido amine) (PAMAM) dendrimer or random coil linear N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer containing free amines in the side chains. The in vitro dark cytotoxicity and phototoxicity of MTCP and related conjugates are assessed on mouth epidermal carcinoma (KB) and human adenocarcinoma alveolar basal epithelial (A549) cells. Phototoxicity of polymeric conjugates increases by ≈100 and 4000 fold in KB and A549 cells compared with nonconjugated MTCP. The increase in phototoxicity activity is shown to result from increased rate of cellular uptake, whereas, cellular internalization of MTCP is negligible in comparison with the conjugated forms. The results of this study suggest the superiority of amine-terminated HPMA copolymer versus PAMAM dendrimer under study for delivery of MTCP. Treatment with various pharmacological inhibitors of endocytosis shows that polymer architecture influences the mechanism of cellular uptake of the conjugated photosensitizer. Results show that polymeric conjugates of MTCP improve solubility, influence the route and the rate of cellular internalization, and drastically enhance the uptake of the photosensitizer.
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Dendrímeros/química , Endocitosis , Metacrilatos/química , Fotoquimioterapia/métodos , Porfirinas/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Dendrímeros/síntesis química , Portadores de Fármacos/química , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Endocitosis/efectos de los fármacos , Citometría de Flujo , Humanos , Metacrilatos/síntesis química , Microscopía Confocal , Nanopartículas/química , Porfirinas/química , Porfirinas/toxicidad , Termodinámica , Factores de TiempoRESUMEN
In photodynamic therapy (PDT) of cancer both the light and the photosensitizing agent are normally harmless, but in combination they could result in selective tumor killing. Zinc oxide nanoparticles were synthesized and coated with the amino acid cysteine to provide an adequate arm for conjugation with porphyrin photosensitizers (meso-tetra (4-carboxyphenyl) porphyrin [MTCP] and CuMTCP). Porphyrin-conjugated nanoparticles were characterized by TEM, FTIR, and UV-vis, and fluorescence spectrophotometry. The 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay was used to measure cell viability in the presence or absence of porphyrin conjugates following UV and X-ray irradiation. The uptake of the porphyrin-conjugated ZnO nanoparticles by cells was detected using fluorescence microscopy. Our results indicated that the survival of T-47D cells was significantly compromised in the presence of ZnO-MTCP-conjugated nanostructures with UV light exposure. Exhibition of cytotoxic activity of ZnO-MTCP for human prostate cancer (Du145) cells occurred at a higher concentration, indicating the more resistant nature of these tumor cells. ZnO-CuMTCP showed milder cytotoxic effects in human breast cancer (T-47D) and no cytotoxic effects in Du145 with UV light exposure, consistent with its lower cytotoxic potency as well as cellular uptake. Surprisingly, none of the ZnO-porphyrin conjugates exhibited cytotoxic effects with X-ray irradiation, whereas ZnO alone exerted cytotoxicity. Thus, ZnO and ZnO-porphyrin nanoparticles with UV or X-ray irradiation may provide a suitable treatment option for various cancers.
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Proliferación Celular/efectos de los fármacos , Cobre/farmacología , Metaloporfirinas/farmacología , Fármacos Fotosensibilizantes/farmacología , Óxido de Zinc/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Cobre/química , Humanos , Metaloporfirinas/química , Nanopartículas/química , Neoplasias/tratamiento farmacológico , Fotoquimioterapia , Fármacos Fotosensibilizantes/química , Rayos Ultravioleta , Rayos X , Óxido de Zinc/químicaRESUMEN
There are a number of reports demonstrating a relationship between the alterations in DFF40 expression and development of some cancers. Here, increased DFF40 expression in T-47D cells in the presence of doxorubicin was envisaged for therapeutic usage. The T-47D cells were transfected with an eukaryotic expression vector encoding the DFF40 cDNA. Following incubation with doxorubicin, propidium iodide (PI) staining was used for cell cycle distribution analysis. The rates of apoptosis were determined by annexin V/PI staining. Apoptosis was also evaluated using the DNA laddering analysis. The viability of DFF40-transfected cells incubated with doxorubicin was significantly decreased compared with control cells. However, there were no substantial changes in the cell cycle distribution of pIRES2-DFF40 cells incubated with doxorubicin compared to control cells. The expression of DFF40, without doxorubicin incubation, had also no significant effect on the cell cycle distribution. There was no DNA laddering in cells transfected with the empty pIRES2 vector when incubated with doxorubicin. In contrast, DNA laddering was observed in DFF40 transfected cells in the presence of doxorubicin after 48 h. Also, the expression of DFF40 and DFF45 was increased in DFF40 transfected cells in the presence of doxorubicin enhancing cell death. Collectively our results indicated that co-treatment of DFF40-transfected cells with doxorubicin can enhance the killing of these tumor cells via apoptosis. Thus, modulation of DFF40 level may be a beneficial strategy for treatment of chemo-resistant cancers.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Desoxirribonucleasas/metabolismo , Doxorrubicina/farmacología , Resistencia a Antineoplásicos , Proteínas de Neoplasias/metabolismo , Inhibidores de Topoisomerasa II/farmacología , Antibióticos Antineoplásicos/farmacología , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Caspasa 3/genética , Caspasa 3/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Inhibición de Migración Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Desoxirribonucleasas/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteínas de Neoplasias/genética , Proteínas de Unión a Poli-ADP-Ribosa , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
Alterations in expression of the DFF40 gene have been reported in some cancers. This study is an in vitro study of the therapeutic effects of gene transfer that lead to elevation in DFF40 expression within T-47D cells in the presence of sulfonamide drugs. In this study, we have constructed a eukaryotic expression vector for DFF40 and transfected it into T-47D cancer cells. We used real time RT-PCR to detect the expression of DFF40 and the MTT assay to determine effects of the sulfonamide drugs acetazolamide, sulfabenzamide, sulfathiazole and sulfacetamide on cell viability in the presence of increased and normal DFF40 levels. Cell cycle distribution was assessed by propidium iodide (PI) staining and the rates of apoptosis by annexin V/PI staining. The DNA laddering analysis was employed to evaluate apoptosis. We observed that overexpression of DFF40 was only effective in decreasing viability in cells incubated with acetazolamide and sulfabenzamide. There was enhanced apoptosis in these groups, particularly with acetazolamide. The cell cycle distribution analysis showed that in the presence of sulfonamide drugs there were no substantial changes in empty-vector or DFF40-transfected cells, except for those cells treated with sulfabenzamide or sulfathiazole. There was no DNA laddering in cells that expressed the empty vector when incubated with sulfonamide drugs. In contrast, we observed DNA laddering in cells that expressed DFF40 in the presence of acetazolamide. Our results have demonstrated that combinatorial use of some sulfonamides such as acetazolamide along with increased expression of DFF40 can potently kill tumor cells via apoptosis and may be beneficial for treatment of some chemoresistant cancers.
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Acetazolamida/farmacología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/genética , Desoxirribonucleasas/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Anexina A5 , Apoptosis/fisiología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , ADN Complementario/genética , Femenino , Vectores Genéticos/genética , Humanos , Proteínas de Unión a Poli-ADP-Ribosa , Propidio , Sulfonamidas/farmacologíaRESUMEN
The inhibitory effects of acetazolamide on the growth and proliferation of epithelial breast cancer cells (T-47D) were investigated. Analysis of morphological changes indicated little apoptosis in T-47D cells incubated with acetazolamide, according to data from flow cytometry, DNA laddering, and expression of AIF. However, an increase in caspase-3 activity was detected in cells. This was concomitant with an increase in DFF45/DFF40 ratio leading to inhibition of caspase-3 activity, DNA fragmentation and progression of apoptosis. Flow cytometry also confirmed that acetazolamide had no significant effect on cell cycle progression. These results are consistent with lack of change in the expression of cell cycle regulatory proteins p21, p27, cdc2 and cyclinD1. Increased expression of ATG5, p53 and DRAM, along with an increase in BCLN1/Bcl-2 ratio, indicated that acetazolamide inhibited the proliferation of T-47D cells by inducing autophagy. Increased expression of PTEN, along with decreased expression of Akt1, also showed that acetazolamide treatment resulted in death inducing autophagy. Collectively the results indicate that autophagy is an adequate mechanism mediating the anti-cancer effects of acetazolamide in T-47D cells through engagement of p53/DRAM pathway and attenuation of Akt survival signalling.
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Acetazolamida/farmacología , Antineoplásicos/farmacología , Autofagia/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Mama/efectos de los fármacos , Inhibidores de Anhidrasa Carbónica/farmacología , Anticonvulsivantes/farmacología , Apoptosis/efectos de los fármacos , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Fragmentación del ADN/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , HumanosRESUMEN
PURPOSE: RNA interference (RNAi) has become a promising tool for cancer therapy. Small interfering RNAs (siRNAs) can synergistically enhance the cell killing effects of drugs used in cancer treatment. Here we examined the effects of siRNA-mediated DNA fragmentation factor 45 (DFF45) gene silencing on breast cancer cell viability, cell cycle arrest, and apoptosis in the presence and absence of doxorubicin. METHODS: We designed three siRNAs, which target different regions of the DFF45 mRNA. Gene silencing was confirmed by real time RT-PCR and Western blot analyses. The impact of DFF45 siRNA, doxorubicin, and their combination on the viability, cell cycle and apoptosis of T-47D and MDA-MB-231 breast cancer cells were determined by MTT, PI staining, annexin V binding, caspase-3 activity, DNA laddering, and chromatin condensation assays. RESULTS: Based on flow cytometric analyses, we found that silencing of DFF45 alone had little effect on apoptosis, especially in T-47D cells. However, when used in combination with doxorubicin (0.33 µM) a significant increase (P < 0.05) in apoptosis was observed in T-47D and MDA-MB-231 cells, i.e., ~2.5- and 3-fold, respectively. Caspase-3 activity, chromatin condensation, as well as DNA laddering supported increased apoptosis in the combinatorial treatment. Cell cycle arrest in both cell lines occurred at lower levels after siRNA + doxorubicin treatment compared to doxorubicin only. CONCLUSIONS: Our data indicate that DFF45 gene silencing, when applied in combination with doxorubicin, may offer a novel therapeutic strategy for the treatment of breast cancer.
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Proteínas Reguladoras de la Apoptosis/genética , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Doxorrubicina/farmacología , Interferencia de ARN , Antibióticos Antineoplásicos/farmacología , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Caspasa 3/metabolismo , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Fragmentación del ADN/efectos de los fármacos , Citometría de Flujo , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Nanoparticles of ultra low molecular weight chitosan (ULMWCS)/hyaluronic acid (HA) were prepared by ion gelation. Three independent variables, namely, ratio of concentration of ULMWCS to HA (CS/HA), pH of solution and stirring time were studied to identify their effects on size, polydispersity and zeta potential of prepared nanoparticles using a Box-Behnken design. Results showed that pH and CS/HA have a direct effect on size, while increase of stirring time decreases the size of nanoparticles. Additionally, it was shown that all the independent parameters have direct effects on zeta potential. Also, the minimum polydispersity index was observed at lowest values of CS/HA. The model also predicted that the optimum values are 4.15, 4.14 and 180 (min) for the CS/HA, solution pH and stirring time, respectively. The obtained preparation had a size of 200 nm, polydispersity index of 0.37, and zeta potential of 13.0 mV.
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Quitosano/química , Ácido Hialurónico/química , Nanopartículas/química , Portadores de Fármacos/química , Concentración de Iones de Hidrógeno , Peso Molecular , Nanopartículas/ultraestructura , Tamaño de la PartículaRESUMEN
The broad spectrum of the pharmacological effects of sulphonamide family of drugs motivated us to investigate the cellular mechanisms for anti-cancer effects of sulphathiazole and sulphacetamide on T-47D breast cancer cells. Fluorescent microscopy, flow cytometric analysis, caspase-3 activity and DNA fragmentation assays were used to detect apoptosis. The distribution of the cells among different phases of the cell cycle was measured by flow cytometry. The expression of several genes with important roles in some critical cellular pathways including apoptosis, mTOR/AKT pathway and autophagy were determined by real-time RT-PCR analysis. Sulphathiazole and sulphacetamide induced anti-proliferative effects on T-47D cells were independent of apoptosis and cell cycle arrest. The overexpression of critical genes involved in autophagy including ATG5, p53 and DRAM indicated that the main effect of the drug-induced anti-proliferative effects was through induction of autophagy. This process was induced in two different forms, including death inducing and cytoprotective autophagy. Sulphathiazole treatment was followed by higher expression of p53/DRAM and downregulation of Akt/mTOR pathway resulting in death autophagy. In contrast, sulphacetamide treatment lowered expression of p53/DRAM pathway in parallel with upregulation of Akt/mTOR pathway promoting cytoprotective autophagy. The results indicated that autophagy is the main mechanism mediating the anti-cancer effects of sulphathiazole and sulphacetamide on T-47D cells. Alignment of the p53 and DRAM expression along with activation level of Akt survival pathway therefore determines the type of autophagy that occurs.
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Antibacterianos/farmacología , Autofagia/efectos de los fármacos , Sulfacetamida/farmacología , Sulfatiazoles/farmacología , Apoptosis , Caspasa 3/metabolismo , Ciclo Celular , Línea Celular Tumoral , Citoprotección , Fragmentación del ADN , Activación Enzimática , Humanos , Dosificación Letal Mediana , SulfatiazolRESUMEN
BACKGROUND: The Bcl-2 protein family members have known as essential controllersof mitochondrial pathway of apoptosis. Bax has been implicated as potential tumor suppressor in certain solid tumors such as breast and colorectal carcinoma. DNA methylation of promoter associated CpG islands has known as a common mechanism for gene inactivation in tumor cells. METHODS: The Methylation Specific PCR (MSP) has used to find the methylation profile of the bax gene promoter CpG islands in colorectal and breast cancer cell lines. RESULTS: We have not detected any kind of "CpG islands hypermethylation" in promoter region of the bax gene in T47D, MCF7 (as ER positive), MDA-MB-231 and MDA-MB-468 (as ER negative) breast carcinoma-derived cell lines and colorectal cancer cell lines H29 and Caco II. CONCLUSION: It seems that CpG island methylation could not play the main role in down-regulation of bax gene in breast and colon cancers.
RESUMEN
Widespread use of mobile phones has increased the human exposure to electromagnetic fields (EMFs). It is required to investigate the effect of EMFs on the biological systems. In this paper the effect of mobile phone RF (910MHz and 940 MHz) on structure and function of HbA was investigated. Oxygen affinity was measured by sodium dithionite with UV-vis spectrophotometer. Structural changes were studied by circular dichroism and fluorescence spectroscopy. The results indicated that mobile phone EMFs altered oxygen affinity and tertiary structure of HbA. Furthermore, the decrease of oxygen affinity of HbA corresponded to the EMFs intensity and time of exposure.
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Teléfono Celular , Hemoglobinas/química , Hemoglobinas/metabolismo , Ondas de Radio , Naftalenosulfonatos de Anilina/química , Tampones (Química) , Dicroismo Circular , Fluorescencia , Hemoglobinas/efectos de la radiación , Humanos , Concentración de Iones de Hidrógeno , Oxígeno/metabolismo , Desnaturalización Proteica , Pliegue de Proteína , Estructura Cuaternaria de Proteína , Temperatura , Factores de TiempoRESUMEN
Sulfonamide drugs mediate their main therapeutic effects through modulation of the activity of membrane and cytosolic carbonic anhydrases. How interactions of sulfonamide drugs impact structural properties and activity of carbonic anhydrases requires further study. Here the effect of acetazolamide on the structure and function of bovine carbonic anhydrase II (cytosolic form of the enzyme) was evaluated. The Far-UV CD studies indicated that carbonic anhydrase, for the most part, retains its secondary structure in the presence of acetazolamide. Fluorescence measurements using iodide ions and ANS, along with ASA calculations, revealed that in the presence of acetazolamide minimal conformational changes occurred in the carbonic anhydrase structure. These structural changes, which may involve spatial reorientation of Trp 4 and Trp 190 or some other related aminoacyl residues near the active site, considerably reduced the catalytic activity of the enzyme while its thermal stability was slightly increased. Our binding results indicated that binding of acetazolamide to the protein could occur with a 1:1 ratio, one mole of acetazolamide per one mole of the protein. However, the obtained kinetic results supported the existence of two acetazolamide binding sites on the protein structure. The occupation of each of these binding sites by acetazolamide completely inactivates the enzyme. Advanced analysis of the kinetic results revealed that there are two substrate (p-NPA) binding sites whose simultaneous occupation is required for full enzyme activity. Thus, these studies suggest that the two isoforms of CA II should exist in the medium, each of which contains one substrate binding site (catalytic site) and one acetazolamide binding site. The acetazolamide binding site is equivalent to the catalytic site, thus, inhibiting enzyme activity by a competitive mechanism.
Asunto(s)
Acetazolamida/farmacología , Anhidrasa Carbónica II/antagonistas & inhibidores , Inhibidores de Anhidrasa Carbónica/farmacología , Animales , Anhidrasa Carbónica II/química , Bovinos , Dicroismo Circular , Cinética , Conformación Proteica , Espectrometría de Fluorescencia , Espectrofotometría UltravioletaRESUMEN
Combinations of multiple linear regressions, genetic algorithms and artificial neural networks were utilized to develop models for seeking quantitative structure-activity relationships that correlate structural descriptors and inhibition activity of adenosine deaminase competitive inhibitors. Many quantitative descriptors were generated to express the physicochemical properties of 70 compounds with optimized structures in aqueous solution. Multiple linear regressions were used to linearly select different subsets of descriptors and develop linear models for prediction of log(k(i)). The best subset then fed artificial neural networks to develop nonlinear predictors. A committee of six hybrid models - that included genetic algorithm routines together with neural networks - was also utilized to nonlinearly select most efficient subsets of descriptors in a cross-validation procedure for nonlinear log(k(i)) prediction. The best prediction model was found to be an 8-3-1 artificial neural network which was fed by the most frequently selected descriptors among these subsets. This prediction model resulted in train set root mean sum square error (RMSE) of 0.84 log(k(i)) and prediction set RMSE of 0.85 log(k(i)) (both equivalent of 0.10 in normal range of log(k(i))) and correlation coefficient (r(2)) of 0.91.
Asunto(s)
Inhibidores de la Adenosina Desaminasa , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Modelos Químicos , Adenosina Desaminasa/metabolismo , Algoritmos , Unión Competitiva , Diseño de Fármacos , Humanos , Técnicas In Vitro , Modelos Lineales , Redes Neurales de la Computación , Dinámicas no Lineales , Relación Estructura-Actividad CuantitativaRESUMEN
The electrostatic interaction of amino acid lysines 190, 195 and 199 of human serum albumin (HSA) with bilirubin have been investigated using molecular dynamic simulations, QM and QM/MM minimization methods. In this study two methodological approaches have been employed. In the first approach X-ray structure and the structure obtained from the molecular dynamic simulation of subdomain IIA of HSA in vacuum have been utilized. Interactions have been evaluated with the segment 186-200 of the cited subdomain. Calculations on the X-ray structure of above segment indicate an effective interaction of the lysine 195 with bilirubin, although that of the lysine 190 is also found considerable in this structure. Performing simulation in vacuum, it has been revealed that except for the lysine 195, the other two lysine residues (190 and 199) could not be considered as centers of interaction. Such finding, which is in accord with experimental data, lends support to the procedure employed in this study. NBO analyses suggest that tasks to achieve a structure indicating bilirubin interaction with the lysine 195 from the 186-200 segment extracted from X-ray structure, results in a structure that lacks any electrostatic interaction. In fact, it has been found that the stability of the latter species can be attributed to the H-bonding interaction of the glutamate 188 with both bilirubin and the lysine 195. Further NBO analysis on the structure of the same species, while achieved after molecular dynamic simulation on subdomain IIA in vacuum has revealed that a favorable electrostatic interaction between the lysine 195 and bilirubin has occurred. Besides, H-bonding interaction of the glutamate 188 with bilirubin has been evident in the same species. For the second approach, presence of water molecules and ions has been considered to simulate condensed medium. Applying docking, conformational sampling, and QM/MM minimization steps in sequence, a structure has been achieved which presents a specific interaction between epsilon-NH3(+) group of the lysine 195 residue and the lactam oxygen atom of bilirubin. NBO analyses suggest that above electrostatic interaction is combined with hydrogen bonding interaction between same two groups. Moreover, a hydrogen bond between oxygen atom of bilirubin's acetate group and alpha-NH group of lysine 195 has been observed. Molecular orbital calculations have been presented which support the NBO analyses.