Silica nanoparticles induce multinucleation through activation of PI3K/Akt/GSK-3β pathway and downregulation of chromosomal passenger proteins in L-02 cells

Silica nanoparticles (SNPs) are applicable in various fields due to their unique physicochemical characteristics. However, concerns over their potential adverse effects have been raised. In our previous studies, we reported that SNPs could induce abnormal high incidence of multinucleation. The aim of this study is to further investigate the mechanisms of multinucleation induced by SNPs (68 nm) in human normal liver L-02 cells (L-02 cells). In order to determine the cytotoxicity of SNPs, MTT assay was performed, and the cell viability was decreased in a dose-dependent manner. The intracellular reactive oxygen species (ROS) detected by flow cytometry and multinucleation observed by Giemsa stain showed that ROS generation and rate of multinucleated cells increased after SNPs exposure. N-acetyl-cysteine (NAC), a glutathione precursor against SNP-induced toxicity, was used as a ROS inhibitor to elucidate the relationship between ROS and multinucleation. The presence of NAC resulted in inhibition of both ROS generation and rate of multinucleation. Moreover, Western blot analysis showed that the protein levels of Cdc20, Aurora B, and Survivin were down-regulated, and the PI3K/Akt/GSK-3β pathway was activated by SNPs. In conclusion, our findings strongly suggested that multinucleation induced by SNPs was related to PI3K/Akt/GSK-3β signal pathway activation and downregulation of G2/M phase-related protein and chromosomal passenger proteins.     

Cytoskeleton and Chromosome Damage Leading to Abnormal Mitosis Were Involved in Multinucleated Cells Induced by Silicon Nanoparticles

Recently, amorphous silicon nanoparticles (SNPs) are widely used in a variety of fields, especially in biological and medical science. Thus, the adverse effect of these nanoparticles should be carefully investigated. The multinucleation effect of SNPs was firstly reported in our previous studies, while the relative mechanism is still unclear. Therefore, the purpose of this study is to investigate the mechanisms with regard to the formation of multinucleated cells. Two sizes of amorphous SNPs (Nano‐Si64 and Nano‐Si46) are carefully characterized. Cytotoxicity and rate of multinucleated cells are firstly determined after human hepatic L‐02 cells are treated with two SNPs for 24 h. Then cell fusion and abnormal mitosis, two ways could form multinucleated cells, are investigated, respectively. Results indicated that SNPs produce a dose‐dependent and size‐related multinucleation effect in L‐02 cells. Abnormal mitosis instead of cell fusion is the main reason for the formation of multinucleated cells caused by SNPs. Both two SNPs could affect the quantity and distribution of cytoskeleton through extra ROS and Ca²⁺ leading to abnormal mitosis and cytokinesis. Additionally, chromosome damage resulting in corresponding G2/M cell cycle arrest should be another aspect, which finally leads to the formation of multinucleated cells in L‐02 cell line.     

Specific binding of tubeimoside-2 with proteins in hepatocarcinoma HepG2 cells: investigation by molecular spectroscopy

In this study, we compared different binding interactions of TBMS2 with proteins both in hepatocarcinoma HepG2 cells and in normal embryo hepatic L02 cells by using fluorescence, absorption, and CD spectroscopy. The fluorescence data revealed that the fluorescence intensity of proteins in the HepG2 and L02 cells decreased in the presence of TBMS2 by 30.79% and 12.01%, respectively. Binding constants and thermodynamic parameters were obtained for systems of TBMS2 with the two kinds of cell proteins. The results indicated that HepG2 cell proteins had a higher TBMS2 binding activity than those in the L02 cells. Analysis of the TBMS2 cytotoxic activities showed that TBMS2 could selectively induce apoptosis of HepG2 cells by binding to them, while its apoptotic effect on L02 cells was relatively weaker.     

Cytotoxic effects and in vitro reversal of multidrug resistance by therapeutic ultrasound in human hepatocarcinoma cell line (HepG2)

Multidrug resistance (MDR) is one of the major obstacles to successful chemotherapy of human malignancies. Although many strategies have been explored to overcome MDR, none of them have been proven to be clinically useful until now. The aim of this study was to investigate whether a novel therapeutic ultrasound (US) approach would have useful effects on the reversal of MDR in cancer cells. Wild-type and MDR phenotype (HepG2/ADM) cells of the human hepatocarcinoma cell line HepG2 were exposed to 0.8 MHz US at an intensity of 0.43 W/cm² for a 9 s exposure (total energy density: 3.87 J/cm²). After US exposure, cell number and viability were counted immediately, and flow cytometry was performed to measure retention of rhodamine 123 and adriamycin in HepG2 and HepG2/MDR cells. Both cell lines were then incubated in suspension with adriamycin, vincristine, etoposide, cisplatin and 5-fluorouracil, respectively, and the MTT assay was used to determine cytotoxicity. The results showed that US exposure could significantly increase the uptake of Rh123 and ADM by HepG2/ADM tumor cells. The resistant index for the chemotherapeutic drugs was significantly lower in the US-exposed HepG2/ADM cells than in those not exposed to US. It was therefore concluded that US exposure could enhance the sensitivity of HepG2/ADM tumor cells to these chemotherapeutic agents, and the functional and structural changes induced by previous US exposure in MDR tumor cells may be responsible for it. However, further study is needed to investigate the mechanism behind US-mediated reversal of MDR.     

9.33 GHz高功率脉冲微波对IAR20鼠肝细胞和L-02人肝细胞增殖的影响

研究了9.33 GHz高功率脉冲微波对IAR20鼠肝细胞和L-02人肝细胞增殖的影响,利用噻唑蓝比色法测量细胞增殖并对实验数据进行拟合,得到脉冲个数、场强和脉宽与细胞增殖之间的关系。当脉冲微波场强与脉宽保持不变,脉冲微波细胞效应随脉冲个数呈现非线性的指数递增规律。当脉冲微波的脉冲个数、脉宽一定时,场强越大,细胞增殖被抑制的程度越大;当脉冲个数、场强不变,脉宽越大,细胞增殖受到抑制的作用越明显,即脉冲微波细胞效应与场强和脉宽成正比。相同脉冲微波参量对不同种类细胞增殖的影响是不同的,对IAR20鼠肝细胞的影响比对L-02人肝细胞的影响略大。     

不同LET射线对细胞的生物学效应

以人肝癌细胞系和正常肝细胞系为材料,报道了不同传能线密度射线辐射引发细胞染色体原初断裂及24 h内的修复情况。 计算了相对生物学效应的值。 以L02染色体总断裂数量得出的RBE值96.05 keV/μm的12C6+ 为3.6, 512 keV/μm 36Ar18+ 为2.9。 而以7721染色体总断裂数量得出的RBE值: 96.05 keV/μm的12C6+ 为3.5,512keV/μm 36Ar18+也为2.9。用产生等点染色单体断裂计算,则RBE更高。对比得出,高LET对增加等点染色单体断裂量的作用要远远大于对增加染色单体断裂量的作用。等点染色单体的断裂修复难度要远远大于染色单体断裂的修复难度, 这也是高LET高致死率的一个重要原因。 Human hepatoma SMMC 7721 and normal liver L02 cells were irradiated with γ rays,12C6+ and 36Ar18+ ion beams at the Heavy Ion Research Facility in Lanzhou(HIRFL). We reported the kinetic repair of chromosome breaks of L02 and SMMC 7721 cells in 24 h of post irradiation time. The relative biological effectiveness(RBE) for inducing chromatid breaks were 3.6 for L02 and 3.5 for SMMC 7721 cell lines at the linear energy transfer(LET) peak of 96.55 keV/μm 12C6+ ions, and 2.9 (both of the two cell lines) at 512 keV/μm 36Ar18+ ions.It suggested that the RBE of isochromatid type breaks induced by 36Ar18+ was higher than those by 12C6+. We concluded that the high production of isochromatid type breaks, induced by the densely ionizing track structure, could be regarded as a signature of high LET radiation exposure.     

Chromatin supraorganization, mitotic abnormalities and proliferation in cells with increased or down-regulated lox expression: Indirect evidence of a LOX-histone H1 interaction in vivo

Lysyl oxidases (LOXs) are enzymes that permit the covalent crosslinking of the component chains of collagen and elastin. These enzymes are present inside the nuclei of certain mammalian cells. Previous studies have proposed LOX binding to histone H1 in vitro, and histone H1 is known to control global chromatin compaction and mitotic chromosome architecture. Therefore, in the present study, we analyzed chromatin supraorganizational changes, mitotic abnormalities, mitotic indices and cell death ratios in COS-7 and NRK-49F cells with high and low lox expression levels, respectively. The objective was to support biochemical data of LOX-H1 interaction, by providing evidence of chromatin remodeling in vivo, under different lox expressions. Chromatin decondensation assessed by image analysis was observed in COS-7 cells with increased lox expression. This decondensation is suggested to be promoted by LOX actions on histone H1, which loosens the DNA-H1 complex. In NRK-49F cells transfected with antisense lox or subjected to treatment with beta-aminopropionitrile (BAPN), chromatin condensation and nuclear phenotypic variability were found, which may be due to reduced LOX-H1 interaction. When lox expression was increased in COS-7 cells, the frequency of irregular chromosome plates was not affected, but cell proliferation decreased and "cell death preceded by multinucleation" increased. In NRK-49F cells there was accelerated proliferation induced by transfection with the antisense lox, and confirmed when cells were treated with BAPN. Apoptosis increased in NRK-49F cells only with BAPN treatment whereas cell death preceded by multinucleation increased only after antisense lox transfection. The data presented herein regarding chromatin remodeling indirectly support the hypothesis that LOX binds to histone H1 in vivo. Cell proliferation in COS-7 and NRK-49F cells and cell death at least in COS-7 cells agree with predicted effects of LOX interference in these processes.     

HLDPE/organic functionalized SiO2 nanocomposites with improved thermal stability and mechanical properties

High-pressure low-density polyethylene (HLDPE)/organic functionalized SiO₂ nanocomposites were synthesized using melt-blending technique in a sigma internal mixer. The properties of the nanocomposites were studied using two different organic functional modifiers: diglycidyl ether of bisphenol-A (DGEBA) and triacetoxyvinylsilane. Reinforcing, thermal stability and toughening effects of organic functionalized nanosilica on the polymer matrix were found at loading of 2.5% nanosilica functionalized with 2.8% of DGEBA and silane coupling agent respectively. Organic functionalization on the nanosilica particle surface led to different microstructures when compared with that of the pure polymer. Organic functionalization on the nanosilica particle surface produced good interfacial adhesion and homogeneous dispersion in the polymer matrix, while the use of nanosilica resulted in aggregated silica particles in the polymer matrix. There was no significant improvement in thermal stability and mechanical properties when only nanosilica was added to the pure polymer. On the contrary, the addition of pretreated nanosilica with organic functional modifiers led to an increase of thermal stability from 313–363°C, elastic modulus and toughness from 0.12–0.18 GPa and 3.23–9.81 MJ/m³ respectively.     

Hypocrellin B-mediated sonodynamic action induces apoptosis of hepatocellular carcinoma cells

Objective

The present study aims to investigate apoptosis of hepatocellular carcinoma cells induced by hypocrellin B-mediated sonodynamic action.

Methods

The hypocrellin B concentration was kept constant at 2.5 μM and cells from the hepatocellular carcinoma HepG2 cell line were exposed to ultrasound with an intensity of 0.46 W/cm² for 8 s. Cell cytotoxicity was quantified using an MTT assay 24 h after sonodynamic therapy (SDT) of hypocrellin B. Apoptosis was investigated using a flow cytometry with Annexin V-FITC and propidium iodine staining. Intracellular reactive oxygen species (ROS) levels were detected using a flow cytometry with 2,7-dichlorodihydrofluorecein diacetate (DCFH-DA) staining.

Results

The cytotoxicity of hypocrellin B-mediated sonodynamic action on HepG2 cells was significantly higher than those of other treatments including ultrasound alone, hypocrellin B alone and sham treatment. Flow cytometry showed that hypocrellin B-induced sonodynamic action markedly enhanced the apoptotic rate of HepG2 cells. Increased ROS was observed in HepG2 cells after being treated with hypocrellin B-mediated sonodynamic action.

Conclusions

Our data demonstrated that hypocrellin B-mediated sonodynamic action remarkably induced apoptosis of HepG2 cells, suggesting that apoptosis is an important mechanism of cell death induced by hypocrellin B-mediated SDT.     

Fluorescence and HPLC Detection of Hydroxyl Radical by a Rhodamine-Nitroxide Probe and its Application in Cell Imaging

A rhodamine nitroxide probe was designed to detect the hydroxyl radical (·OH), which presented high selectivity for ·OH over other reactive oxygen species (ROS) and linear fluorescence response to ·OH produced by Fenton reaction. The product was detected by HPLC-MS, indicating that the main product of the reaction was O-methylhydroxylamine and the product peak areas measured by HPLC-UV/vis and HPLC-FLD both enhanced proportionally with the increase of ·OH concentration. The application of the probe in biological system was explored to trace the production of ·OH in cells under oxidative stress condition induced by rotenone which can inhibit the mitochondria respiratory chain complex I and we found that appropriate rotenone may induce the normal human liver cells (L02) and human hepatoma cells (HepG2) to produce ·OH at different degrees.     

灵芝酸A对人肝癌细胞的辐射增敏效应的初步研究

本研究旨在初步探讨灵芝酸A(GAA)对人肝癌细胞系HepG2在高LET中子和低LET的γ射线条件下的辐射敏感性的影响及差异。研究中,我们用CCK-8方法检测不同浓度GAA对HepG2增殖抑制作用。选取低浓度(5μmol/L)GAA预处理细胞24 h,分别给予不同剂量的中子辐照或γ射线辐照,分别检测克隆存活率、细胞凋亡和γH2AX蛋白的foci的形成。结果表明:在不加GAA的情况下,高LET中子辐射比低LET的γ射线对细胞产生的凋亡比例高;在添加了GAA后,与未加GAA对照组相比,诱导细胞凋亡的比例明显增加;另外,加GAA处理后,细胞增殖抑制率也随着辐照剂量的增加而增高。即GAA能增加HepG2细胞的辐射敏感性,而在同样GAA剂量下,HepG2细胞对高LET中子辐射比低LET的γ射线更敏感。由此,这项研究说明灵芝酸或可开发成为一种天然辐射增敏剂,从而为癌症特别是肝癌的放疗提供新的辅助治疗方法。     

Influence of particle size on the low and high strain rate behavior of dense colloidal dispersions of nanosilica

Shear thickening is a non-Newtonian flow behavior characterized by the increase in apparent viscosity with the increase in applied shear rate, particularly when the shear rate exceeds a critical value termed as the critical shear rate (CSR). Due to this remarkable property of shear-thickening fluids (STFs), they are extensively used in hip protection pads, protective gear for athletes, and more recently in body armor. The use of STFs in body armor has led to the development of the concept of liquid body armor. In this study, the effect of particle size is explored on the low and high strain rate behavior of nanosilica dispersions, so as to predict the efficacy of STF-aided personal protection systems (PPS), specifically for ballistic applications. The low strain rate study was conducted on cone and plate rheometer, whereas the high strain rate characterization of STF was conducted on in-house fabricated split Hopkinson pressure bar (SHPB) system. Spherical nanosilica particles of three different sizes (100, 300, and 500 nm) as well as fumed silica particles of four different specific surface areas (Aerosil A-90, A-130, A-150, and A-200), respectively, were used in this study. The test samples were prepared by dispersing nanosilica particles in polypropylene glycol (PPG) using ultrasonic homogenization method. The low strain rate studies aided in determining the CSR of the synthesized STF dispersions, whereas the high strain rate studies explored the impact-resisting ability of STFs in terms of the impact toughness and the peak stress attained during the impact loading of STF in SHPB testing.     

Ultrasound-induced cytolysis of cancer cells is enhanced in the presence of micron-sized alumina particles

Micron-sized alumina particles have been shown to enhance sonochemical free radical formation in aqueous solutions and simultaneously increase the solution temperature and acoustic (white) noise, effects attributable to enhanced inertial cavitation [T. Tuziuti, J. Phys. Chem. A 109 (2005) 4869–4872]. In the current study, the same ultrasound exposure system was applied to in vitro cancer cells as a model system to determine the effect of alumina particles on the long-term survival of cells and on the major pathways of cell death, i.e., either apoptosis or necrosis. Following 6 h of incubation after ultrasound treatment, it was found that the cells died mainly through necrosis, irrespective of whether the exposure was conducted in the presence of alumina particles or not. Alumina particles were non-toxic to cells alone, but were found to decrease the long-term survivability of cells that survived the initial exposure. This effect depended on the size and concentration of particles. These results correlated well with the effect of alumina particles on the sonochemical oxidation of KI under the same exposure conditions. Spin-trapping with 5,5-dimethyl-pyroline N-oxide (DMPO) and electron spin resonance spectroscopy indicated that the sonochemical formation of OH radicals increased in the presence of alumina particles. The current study is consistent with the well known observation that micron-sized particles enhance the acoustic cavitation process.     

γ射线对肝癌细胞G2染色体的原初损伤

用Calyculin—A诱导的早熟染色体凝集技术研究了γ射线诱导人肝癌细胞株HepG2细胞G2期染色体的原初损伤。结果表明:G2等点染色单体断裂畸变与辐射剂量呈线性平方关系,G2染色单体断裂畸变和G2期染色单体断裂畸变总数与辐射剂量呈线性正相关关系,发生各类断裂畸变的细胞率与剂量也呈线性正相关关系。γ射线诱发的断裂畸变主要是G2染色单体断裂畸变,断裂畸变的细胞主要是发生G2染色单体断裂畸变。A chemically induced premature chromosome condensation technique with Calyculin-A has been employed to estimate the initial chromosome damage in HepG2 condensed in G2 phase and the percentage of aberrant cells after exposure to γ-rays. The results show that the dose-response for iso-chromatid breaks is linear-quadratic manner, while chromatid-type breaks and total chromatid breaks show a positive linear dose-response. The percent tages of all kinds of aberrant cells are increasing linearly with increasing doses. G2 chromatid-type breaks and the percentage of G2 chromatid-type aberrant cells are predominate in G2 total chromatid breaks induced b y γ-rays.     

Comparing the Effect of Micro- and Nano-Scale Silica on the Rheological,Dynamic Mechanical Properties and Flame Resistance of Nylon 6,6

Nylon 6,6 micro- and nano-silica composites were prepared by melt processing using a twin-screw extruder. Three nanocomposites containing 4, 8, and 12 wt.% of nanosilica were prepared. In order to compare the effect of size, a microcomposite containing 4 wt.% of micron-size silica was also prepared. The effects of particle type (micro- and nano-size) on the dynamic thermomechanical and rheological properties, morphology, and flame resistance of the composites were examined. The dynamic thermomechanical properties (DMTA), scanning electron microscopy (SEM), transmission electron microscopy (TEM), dynamic rheometry, thermogravimetry analysis (TGA), and limiting oxygen index (LOI) data are reported. The particles were observed to be dispersed uniformly, but with a different level of coalescence, by means of SEM and TEM. The DMTA results showed that the damping factor peak positions of the nanocomposites at low content of nanofiller shifted more to higher temperature compared to those of nanocomposites containing high concentrations of nanofiller. Dynamic rheometry, using a parallel plate rheometer, showed that the rheological moduli of the nanocomposites increased with increase in nanofiller concentration; however, this increase was greater in the high-frequency region. These results showed that increasing the concentration of nanofiller, and the consequent coalescence effect within the nanocomposites, led to rheological moduli values similar to those of the microcomposite. The TGA and LOI results of the microcomposite and nanocomposite containing 4wt.% of nanosilica showed that nanosilica had a more significant effect to enhance the heat and flame resistance of nylon 6,6 compared to that of micron-sized silica.     

Nonisothermal Crystallization Kinetics of Poly(lactic acid)/Nanosilica Composites

Poly(lactic acid) (PLA)/nanosilica composites were prepared by blending the PLA and nanosilica in chloroform and then evaporating the solvent to form the composite films in a dish. The Ozawa and Mo equations were used to characterize the nonisothermal cold crystallization kinetics of the PLA/nanosilica composites. The results indicated that the Ozawa equation was not successful while the Mo equation was successful to describe the nonisothermal crystallization kinetics of PLA/nanosilica composites. The values of crystallization activation energy (E _c) of the samples were calculated by the Kissinger method. Although the sample crystallization rates were enhanced with the increase of nanosilica content, the samples exhibited increased E _c in the presence of nanosilica. The results showed that nanosilica had an effect on both the nucleation and the crystal growth of PLA, promoting the nucleation but interfering with the molecular motion of PLA in the crystallization process.     

Evaluation of Stability and Sensitivity of Cell Fluorescent Labels When Used for Cell Migration

The directed migration of mammalian cells is a foundation of development and growth. A variety of processes such as tissue development, wound healing, pathogen recognition/destruction as well as cancer metastasis are the result of regulated or dysregulated cell migration. While the ability to measure a cell’s propensity to migrate has clinical relevance in several settings, no universal protocol has been established to measure cell migration. A variety of techniques are currently used to measure migration including manual counting, flow cytometry or Coulter counting, microfluidic devices, computerized spectroscopic methods, or the use of various tracking dyes interfaced with fluorescent or non-fluorescent plate readers. In order to expedite the measurement of migration, we compared several common cytoplasmic and lipophilic cell tracking dyes to determine the best dye for determining migration of rare population of cells. CellVue® Burgundy was found to be superior over calcein AM, Cell Tracker Green CMFDA (chloromethyl fluorescein diacetate), Vybrant CFDA (carboxy fluorescein diacetate succinimidyl ester) in its retention within cells, superior to CellVue® NIR 815, PKH67, and CM DiI with regard to signal to noise ratio, and superior to PKH26 with regard to instrument versatility.     

Differential toxicity of amorphous silica nanoparticles toward phagocytic and epithelial cells

The objective of this study was to evaluate the influence of size and surface functionality of amorphous silica nanoparticles (SNPs) on their interaction with cultured cells. The intracellular uptake, phagocytic activity, and possible mechanisms of toxicity induced by SNPs were studied on murine alveolar macrophages and two epithelial cancer cell lines. It was found that phagocytic cells are more susceptible to amorphous SNPs than epithelial cells. SNPs with functionalized surfaces were capable to induce the formation of apoptotic cells to a higher extent than plain particles. Plain SNPs induced plasma membrane damage in phagocytic cells to a higher extent and caused cell death in a shorter period of time than surface-functionalized SNPs. The prevalence of necrotic mode of cell death was observed after treatment with plain SNPs. In the range studied surface functionality played an important role in SNPs toxicity.     

Direct visualization of neo-vessel formation following peripheral injection of bone marrow derived CD34+ cells in experimental myocardial damage

The definitive fate of peripherally injected PKH26 labelled bone marrow mononuclear cells expressing the CD34+ antigen following experimental myocardial cryodamage in rats (n=10) has been examined by direct visualization on photoconverted light and electron microscopy images. One week after the injection in each rat of about 150,000 CD34+ cells early stage PKH26+ vascular structures were localized in the infarcted areas, suggesting that a potential benefit of this therapeutic approach consists in the regeneration of the vasculature.     

Development of a Liver‐Targeting Gold–PEG–Galactose Nanoparticle Platform and a Structure–Function Study

For the specific liver parenchymal cell delivery, a series of short heterobifunctional poly(ethylene glycol) (PEG) derivatives containing dimercapto and galactose (Gal) terminals is synthesized for the preparation of gold conjugates. The Gal density on the surface of all gold conjugates can be well controlled and the prepared gold conjugates are stable in various media, even in the presence of serum. For the liver targeting and reflectance imaging applications, the structure–function relationships of this platform, including the influence of the PEG molecular weight and the Gal ligand coverage of hybrid particles on the cytotoxicity and cellular recognition of tumor cells in vitro and on their liver‐targeting ability in small animals, are studied. Biocompatibility results show that HepG2 cells are more sensitive than HeLa cells to gold conjugates. Cellular uptake studies demonstrate that a lower PEG molecular weight, a higher Gal density, or a higher gold concentration can increase the cellular uptake efficiency of these hybrid particles in HepG2 cells when the other parameters are constant. The results reveal the importance of parameter modulation for the design and control of nanoprobes and the gold conjugates with short PEG chains and a high Gal density are a potential vector for active‐targeting therapy.