Gold nanoparticles conjugated to bimetallic manganese(II) and iron(II) Prussian Blue analogues for aptamer-based impedimetric determination of the human epidermal growth factor receptor-2 and living MCF-7 cells.

An aptamer-based assay is described for the determination of trace levels of the cancer marker human epidermal growth factor receptor-2 (HER2) and living MCF-7 cells. The method is based on the use of a bimetallic MnFe Prussian blue analogue coupled to gold nanoparticles (MnFePBA@AuNP). Compared to pristine MnFe PBA nanocubes, the series of MnFePBA@AuNP exhibits a core-shell spherical nanostructure, and the shell thickness decreases from 99.9 nm down to 49.3 nm on increasing the fraction of AuNPs. The composite was placed on a gold electrode and incubated with the aptamer solution through electrostatic interaction. Then the modified electrode was employed to detect HER2 and MCF-7 cells using [Fe(CN)6]3-/4- as redox probe and displays good responses to both of them. Electrochemical impedance spectroscopy data show that the signal variation between each step during the whole procedure for the HER2 and MCF-7 cells detection can be embodied as the resistance value change between the [Fe(CN)6]3-/4- and electrode surface. The assay has a very low detection limit (0.247 pg∙mL-1) and works in the 0.001-1.0 ng∙mL-1 HER2 concentration range. It was also used to sense HER2 in MCF-7 cells, and this results in an assay that works within the 500-5 × 104 cell∙mL-1 cell concentration range and a 36 cell∙mL-1 detection limit. Furthermore, the aptamer-based assay is selective, acceptably reproducible, stable, and well feasible for the detection of HER2 and living MCF-7 cells in human serum. Graphical abstract Schematic of an electrochemical aptasensor based on the bimetallic MnFe Prussian blue analogue (MnFe PBA) coupling with gold nanoparticles (represented by MnFePBA@AuNPs). It was employed as the aptasensor for human epidermal growth factor receptor-2 (HER2), and living MCF-7 cells.

Characteristics of human adipose mesenchymal stem cells isolated from healthy and cancer affected people and their interactions with human breast cancer cell line MCF-7 in vitro.

Adipose tissue is an attractive source of mesenchymal stem/stromal cells (MSCs) with potential applications in reconstructive plastic surgery and regenerative medicine. The aim of this study was to characterise human adipose tissue MSCs (ASCs) derived from healthy individuals and cancer patients and to compare their interactions with tumour cells. ASCs were isolated from adipose tissue of healthy donors, breast cancer-adjacent adipose tissue of breast cancer patients and tumour-adjacent adipose tissue of non-breast cancer patients. Their proliferation, differentiation, immunophenotype and gene expression were assessed and effects on the proliferation of human breast cancer cell line MCF-7 compared. ASCs from all sources exhibited similar morphology, proliferative and differentiation potential, showing the characteristic pattern of mesenchymal surface markers expression (CD90, CD105, CD44H, CD73) and the lack of HLA-DR and hematopoietic markers (CD11a, CD33, CD45, Glycophorin-CD235a), but uneven expression of CD34. ASCs also shared a common positive gene expression of HLA-DR, HLA-A, IL-6, TGF-ß and HIF-1, but were negative for HLA-G, while the expression levels of Cox-2 and IDO-1 varied. All ASCs significantly stimulated the proliferation of MCF-7 tumour cells in direct mixed co-cultures and transwell system, although their conditioned media displayed antiproliferative activity. Data obtained showed that ASCs with similar characteristics are easily isolated from various donors and sites of origin, although ASCs could both suppress and favour tumour cells growth, emphasising the importance of cellular context within the microenvironment and pointing to the significance of safety studies to exclude any potential clinical risk of their application in regenerative medicine.

[Comparison of cell elasticity analysis methods based on atomic force microscopy indentation].

In order to investigate in greater detail the two methods based on Hertz model for analyzing force-distance curve obtained by atomic force microscopy, we acquired the force-distance curves of Hela and MCF-7 cells by atomic force microscopy (AFM) indentation in this study. After the determination of contact point, Young's modulus in different indentation depth were calculated with two analysis methods of "two point" and "slope fitting". The results showed that the Young's modulus of Hela cell was higher than that of MCF-7 cell,which is in accordance with the F-actin distribution of the two types of cell. We found that the Young's modulus of the cells was decreased with increasing indentation depth and the curve trends by "slope fitting". This indicated that the "slope fitting" method could reduce the error caused by the miscalculation of contact point. The purpose of this study was to provide a guidance for researcher to choose an appropriate method for analyzing AFM indentation force-distance curve.

Poly(iohexol) nanoparticles as contrast agents for in vivo X-ray computed tomography imaging.

Biocompatible poly(iohexol) nanoparticles, prepared through cross-linking of iohexol and hexamethylene diisocyanate followed by coprecipitation of the resulting cross-linked polymer with mPEG-polylactide, were utilized as contrast agents for in vivo X-ray computed tomography (CT) imaging. Compared to conventional small-molecule contrast agents, poly(iohexol) nanoparticles exhibited substantially protracted retention within the tumor bed and a 36-fold increase in CT contrast 4 h post injection, which makes it possible to acquire CT images with improved diagnosis accuracy over a broad time frame without multiple administrations.

Transfer of Ser7 phosphorylated CENP-A from centromere to midbody during mitosis in MCF-7 cells.

Serine 7 of centromere protein A (CENP-A) is a very important mitosis-specific phosphorylation site. In this study, we demonstrate the subcellular distribution of Ser7 phosphorylated CENP-A during mitosis in MCF-7 cells. The Ser7 phosphorylation of CENP-A was observed beginning at prophase at centromeres. Upon progression of mitosis, the fluorescence signals emerged in the central region of the metaphase plate and were maintained until anaphase at centromeres. At late anaphase, the fluorescence signals moved to the midzone gradually and transferred from the centromere to the midbody completely at telophase. They were compacted into the centre of the midbody in a thin cylinder consisting of a sandglass-like "mitotic machine" with microtubules and condensed chromosome. We also found that Ser10 phosphorylated H3 and Thr11 phosphorylated H3 were co-localized at the midbody in two bell-like symmetrical bodies with Ser7 phosphorylated CENP-A during the terminal stage of cytokinesis. Midbody isolation and immunoblotting experiments also indicated that Ser7 phosphorylated CENP-A are components of the midbody. These findings suggest that Ser7 phosphorylated CENP-A acts as a chromosomal passenger protein and may play an important role in cytokinesis.

The Phenotypic Effects of Exosomes Secreted from Distinct Cellular Sources: a Comparative Study Based on miRNA Composition.

Exosomes are nano-sized vesicles composed of lipids, proteins, and nucleic acids. Their molecular landscape is diverse, and exosomes derived from different cell types have distinct biological activities. Since exosomes are now being utilized as delivery vehicles for exogenous therapeutic cargoes, their intrinsic properties and biological effects must be understood. We performed miRNA profiling and found substantial differences in the miRNA landscape of prostate cancer (PC3) and human embryonic kidney (HEK) 293 exosomes with little correlation in abundance of common miRNAs (R2 = 0.16). Using a systems-level bioinformatics approach, the most abundant miRNAs in PC3 exosomes but not HEK exosomes were predicted to significantly modulate integrin signaling, with integrin-ß3 loss inducing macrophage M2 polarization. PC3 but not HEK exosomes downregulated integrin-ß3 expression levels by 70%. There was a dose-dependent polarization of RAW 264.7 macrophages toward an M2 phenotype when treated with PC3-derived exosomes but not HEK-derived exosomes. Conversely, HEK exosomes, widely utilized as delivery vehicles, were predicted to target cadherin signaling, with experimental validation showing a significant increase in the migratory potential of MCF7 breast cancer cells treated with HEK exosomes. Even widely utilized exosomes are unlikely to be inert, and their intrinsic activity ought to be assessed before therapeutic deployment.

Effects of temperature and cellular interactions on the mechanics and morphology of human cancer cells investigated by atomic force microscopy.

Cell mechanics plays an important role in cellular physiological activities. Recent studies have shown that cellular mechanical properties are novel biomarkers for indicating the cell states. In this article, temperature-controllable atomic force microscopy (AFM) was applied to quantitatively investigate the effects of temperature and cellular interactions on the mechanics and morphology of human cancer cells. First, AFM indenting experiments were performed on six types of human cells to investigate the changes of cellular Young's modulus at different temperatures and the results showed that the mechanical responses to the changes of temperature were variable for different types of cancer cells. Second, AFM imaging experiments were performed to observe the morphological changes in living cells at different temperatures and the results showed the significant changes of cell morphology caused by the alterations of temperature. Finally, by co-culturing human cancer cells with human immune cells, the mechanical and morphological changes in cancer cells were investigated. The results showed that the co-culture of cancer cells and immune cells could cause the distinct mechanical changes in cancer cells, but no significant morphological differences were observed. The experimental results improved our understanding of the effects of temperature and cellular interactions on the mechanics and morphology of cancer cells.

Staghorn Sumac Reduces 5-Fluorouracil-Induced Toxicity in Normal Cells.

Edible staghorn sumac (Rhus hirta) fruit extract was applied in conjunction with chemotherapeutic drug 5-fluorouracil to promote cytotoxicity of the drugs toward human breast cancer cells MCF-7 while protecting normal cells MCF-10A from drug toxicity. Sumac extract would be a promising chemotherapeutic drug conjugate in cancer chemotherapy.

Scattering of MCF7 cells by heregulin ß-1 depends on the MEK and p38 MAP kinase pathway.

Heregulin (HRG) ß1 signaling promotes scattering of MCF7 cells by inducing breakdown of adherens and tight junctions. Here, we show that stimulation with HRG-ß1 causes the F-actin backbone of junctions to destabilize prior to the loss of adherent proteins and scattering of the cells. The adherent proteins dissociate and translocate from cell-cell junctions to the cytosol. Moreover, using inhibitors we show that the MEK1 pathway is required for the disappearance of F-actin from junctions and p38 MAP kinase activity is essential for scattering of the cells. Upon treatment with a p38 MAP kinase inhibitor, adherens junction complexes immediately reassemble, most likely in the cytoplasm, and move to the plasma membrane in cells dissociated by HRG-ß1 stimulation. Subsequently, tight junction complexes form, most likely in the cytoplasm, and move to the plasma membrane. Thus, the p38 MAP kinase inhibitor causes a re-aggregation of scattered cells, even in the presence of HRG-ß1. These results suggest that p38 MAP kinase signaling to adherens junction proteins regulates cell aggregation, providing a novel understanding of the regulation of cell-cell adhesion.

Efeitos citotóxicos, genotóxicos e epigenéticos do Bisfenol A em células HL-60, MCF-7 e em ratos / Cytotoxic, genotoxic and epigenetics effects of Bisphenol A on HL-60, MCF-7 cells and rats

Bisfenol A (BPA) é um insumo largamente utilizado na produção de plástico policarbonato e amplamente difundido no meio ambiente, levando o ser humano à exposição crônica desde o período intrauterino. A literatura aponta a possibilidade de BPA aumentar o risco de diversos tipos de câncer, mas são necessários estudos que possibilitem o entendimento de mecanismos pelos quais isso pode ocorrer. Neste trabalho foram investigados os efeitos do BPA ou nitro-BPA em células HL-60, MCF-7 e tecidos de ratos. Células HL-60 foram expostas ao BPA ou nitro-BPA nas concentrações de 25, 100 e 250 µM (0,1 % DMSO v/v) por 2, 24 ou 48 horas na presença ou ausência de H2O2 (40 nmol/5 x 104 células). Células MCF-7 foram expostas da mesma forma, sem o uso de H2O2, mas na presença e ausência de agonista (PCB) de receptor Ah. Ratos Sprague-Dawley machos receberam BPA diariamente ao longo de 4 semanas (50 mg/kg de peso corpóreo) por gavagem, na vigência e ausência de diabetes, com subsequente coleta de urina, fígado, rins, medula óssea e sangue. Nos experimentos com as células, a viabilidade, ciclo celular, fragmentação do DNA e a produção intracelular de espécies reativas de oxigênio (ROs) foram avaliadas por citometria de fluxo, a atividade da cadeia respiratória mitocondrial pelo ensaio do XTT, e a atividade de MPO de células HL-60 por ensaio de fluorescência, bem como a produção de •NO. A metilação e hidroximetilação global do DNA e os adutos 8-oxodG, CEdG, 1,N6-εdA, 1,N2-εdG e BPA-Gua no DNA das células, tecidos, meio de cultura e urina foram analisados por HPLC-ESI-MS/MS. O hemograma e mielograma dos animais foram obtidos no Laboratório de Hematologia Experimental da FCF USP. Observou-se que tanto BPA quanto nitro-BPA induziram a geração de ROS em células HL-60 logo após 2h de incubação. BPA levou subsequentemente à perda de atividade da cadeia respiratória mitocondrial, aumento da permeabilidade da membrana plasmática, fragmentação do DNA, parada na fase G2/M do ciclo celular e hipermetilação acompanhada de hipohidroximetilação global do DNA. A citotoxicidade induzida pelas mesmas concentrações de nitro-BPA em células HL-60 foi menos pronunciada, sem perda de atividade da cadeia respiratória mitocondrial, com pouca fragmentação do DNA, mas com parada na fase G0/G1 do ciclo celular e indução de hipohidroximetilação global do DNA na presença de H2O2. Não foi observada a indução de adutos de DNA nas células HL-60 incubadas com BPA, mas sim de CEdG nas células incubadas com nitro-BPA. Os dados obtidos a partir da exposição das células HL-60 a BPA e nitro-BPA nos indicam que as duas moléculas provocam alterações metabólicas distintas nesse tipo celular, independentes da via estrogênica, que levam a alterações predominantemente epigenéticas (BPA) ou genéticas e epigenéticas (nitro-BPA), que podem ter consequências fenotípicas, como progressão maligna, que precisam ser investigadas. Foi observado que as células MCF-7 são mais resistentes que as células HL-60 à citotoxicidade induzida por BPA e nitro-BPA. Como resultado da exposição das células MCF-7 a BPA, houve pequeno aumento da permeabilidade da membrana plasmática (250 µM), indução dos níveis de ROS após 24 h (25 µM) e aumento da população de células em sub G1, ou seja, com DNA fragmentado (100 µM e 250 µM), mas sem alteração do ciclo celular. No caso de nitro-BPA, foi observada parada do ciclo celular em G2/M (25 µM, 100 µM e 250 µM), assim como aumento de permeabilidade da membrana plasmática após 24 h de incubação (25 µM, 250 µM), sem indução de ROS ou aumento de células em sub G1. Entretanto, observou-se aumento dos níveis de CEdG e 8-oxodG no DNA das células incubadas com BPA (100 µM, 250 µM) sem a ativação prévia de receptores Ah. A ativação dos receptores Ah com PCB levou a menor aumento do nível das lesões após as incubações com BPA. A maior resistência das células MCF-7 aos efeitos citotóxicos do BPA está provavelmente relacionada à ação estrogênica desse xenobiótico. A sinalização estrogênica juntamente com o aumento dos níveis de lesões no DNA aumenta a chance de mutações e de transformação maligna. Nas células com ativação do receptor Ah, BPA levou ainda ao aumento da hidroximetilação global, sem alteração da metilação global do DNA. Os animais não diabéticos expostos ao BPA apresentaram quantidades diminuídas de promielócitos, blastos e bastonetes na medula óssea (aplasia medular), sem alteração no hemograma. Houve aumento dos níveis de CEdG no fígado, da metilação e hidroximetilação global do DNA hepático, e não foi observada alteração das marcas epigenéticas e adutos de DNA no rim ou na urina. Os animais diabéticos expostos ao BPA apresentaram aumento do número de eosinófilos e linfócitos na medula óssea, podendo-se sugerir a indução de um estado inflamatório alérgico, e aumento do número total de hemácias circulantes e do hematócrito. Houve aumento dos níveis de CEdG, da metilação e hidroximetilação global do DNA hepático, aumento dos níveis de 8-oxodG no DNA renal, sem alteração das marcas epigenéticas no rim, e não foi observada alteração dos adutos de DNA na urina. Os dados obtidos apontam para a geração de ROS como uma importante via de cito- e genotoxicidade induzidas por BPA. Sua biotransformação para BPA-3,4- quinona nos modelos utilizados parece ter menor importância para os efeitos, uma vez que não foi detectada a lesão BPA-Gua em nenhuma amostra de DNA, meio de cultura das células ou urina dos animais. Alterações metabólicas induzidas por BPA e ROS podem favorecer as alterações das marcas epigenéticas observadas no DNA das células HL-60, MCF-7 e fígado dos animais. Todas essas alterações podem contribuir para a transformação maligna de células expostas ao BPA

Bisphenol A (BPA) is a compound widely used in polycarbonate plastic production and widespread in the environment, humans are chronic exposed to BPA in intrauterine period and entire life. The literature suggests the possibility of BPA increase the risk of developing cancers, but studies are required to enable the understanding of mechanisms by which this can occur. HL -60 cells were exposed to BPA or nitro-BPA at concentrations of 25, 100 and 250 uM (0.1% DMSO v/v) for 2, 24 or 48 hours in presence or absence of H2O2 (40 nmol/5x104 cells), MCF-7 cells followed a similar profile of exposure without the use of H2O2, but in presence or absence of Ah agonist receptor (PCB126). Male Sprague-Dawley rats received BPA daily over 4 weeks (50 mg/kg body weight) by gavage in presence and absence of diabetes, with subsequent collection of urine, liver, kidney, bone marrow and circulating blood. The viability, cell cycle, DNA fragmentation and the intracellular production of reactive oxygen species (ROS) was evaluated by flow cytometry, MPO activity and NO production was evaluated by fluorescence assay for HL- 60 cells, mitochondrial activity by XTT assay, and the global DNA methylation was checked by HPLC-PDA. DNA adducts 8-oxodG, CEdG, 1,N6-εdA, 1,N6-εdG and BPA-Gua were quantified by HPLC- ESI-MS/MS in DNA of cells, culture medium, urine and tissue collected from Sprague-Dawley rats. Blood count and bone marrow examination were obtained in collaboration with Experimental Hematology Laboratory of University of Sao Paulo We observed that both BPA and BPANO2 induced ROS generation in HL- 60 cells after 2 hours of incubation. BPA subsequently led to failure of mitochondrial respiratory chain activity, increased permeability of the plasma membrane, DNA fragmentation, arrest in G2/M phase of cell cycle, DNA hypermethylation with global hipohydroxymethylation. We saw low cytotoxicity in HL-60 cells induced by nitro-bpa n the same concentration, without loss of mitochondrial respiratory chain activity, discrete DNA fragmentation, but leading cell cycle to stopping at G0/G1 phase, and induction of DNA global hypermethylation. No lesions were observed in the DNA of HL-60 cells.The results obtained from the exposure of HL-60 cells to BPA and nitro-BPA indicate that these two molecules induce different metabolic abnormalities in this cell line, independent of estrogen pathway, leading to changes in epigenetic (BPA) or genetic and epigenetic (nitro-BPA) profile, that can induce phenotypic consequences such as malignant progression. It was observed along the study that MCF-7 cells are more resistant than HL-60 cells to cell damage induced by BPA and nitro-BPA. As a result of MCF-7 cells exposure to BPA, we saw a slight increase in membrane permeability (250 mM), ROS generation after 24h (25 mM) and increase in cell population in sub G1, so we had DNA fragmentation (100 uM and 250 uM), but with no effect on cell cycle. However, we observed increased levels of CEdG and 8-oxodG on DNA of cells incubated with BPA (100 uM, 250 mM) without prior activation of Ah receptors. The activation of Ah receptors with PCB took a small increase in the level of DNA lesions after incubations with BPA. MCF-7 cells resistance to the cytotoxic effects of BPA is probably related to estrogen action of this compound. Estrogen signaling in addition with the increased levels of DNA damage increases the chance of mutations and malignant transformation. In cells with Ah receptor activation, BPA also led to increased DNA global hydroxymethylation, without changing the global DNA methylation. Nondiabetic animals exposed to BPA had decreased amounts of promyelocytes, blasts and rods in the bone marrow, with any change in blood count. There were an increase of CEdG levels in liver, methylation and global hydroxymethylation on hepatic DNA, and was observed any alteration on epigenetic markers and DNA adducts in kidney or urine. On the other hand diabetic animals exposed to BPA showed increased numbers of eosinophils and lymphocytes in bone marrow, suggesting the induction of an allergic inflammatory state. There were increased levels of CEdG, methylation and global hydroxymethylation on hepatic DNA, increased 8-oxodG levels on kidney DNA without changing epigenetic markers, was not observed DNA adducts in urine. The data obtained indicate that the generation of ROS could be the major route of cytotoxic and genotoxic induced by BPA exposure. BPA biotransformation to BPA-3,4-quinone used in the models seem to have poor effects, since we was not detected BPA-Gua lesion in any DNA sample, culture medium of cells or urine of the animals. Metabolic changes induced by BPA and ROS can enable changes in epigenetic markers observed in the DNA of HL-60 cells, MCF-7 and liver tissue. All these changes may contribute to malignant transformation of cells that were exposed to BPA