Aberrant Cytokinesis and Cell Fusion Result in ... - ACS Publications

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Aberrant Cytokinesis and Cell Fusion Result in Multinucleation in HepG2 Cells Exposed to Silica Nanoparticles Yongbo Yu,†,‡ Junchao Duan,†,‡ Weijia Geng,†,‡ Qiuling Li,†,‡ Lizhen Jiang,†,‡ Yang Li,†,‡ Yang Yu,†,‡ and Zhiwei Sun*,†,‡ †

School of Public Health, Capital Medical University, Beijing 100069, P.R. China Beijing Key Laboratory of Environmental Toxicology, Capital Medical University, Beijing 100069, P.R. China

‡

S Supporting Information *

ABSTRACT: The multinucleation effect of silica nanoparticles (SiNPs) had been determined in our previous studies, but the relative mechanisms of multinucleation and how the multinucleated cells are generated were still not clear. This extensional study was conducted to investigate the mechanisms underlying the formation of multinucleated cells after SiNPs exposure. We first investigated cellular multinucleation, then performed time-lapse confocal imaging to certify whether the multinucleated cells resulted from cell fusion or abnormal cell division. Our results confirmed for the first time that there are three patterns contributing to the SiNPs-induced multinucleation in HepG2 cells: cell fusion, karyokinesis without cytokinesis, and cytokinesis followed by fusion. The chromosomal passenger complex (CPC) deficiency and cell cycle arrest in G1/S and G2/M checkpoints may be responsible for the cell aberrant cytokinesis. The activated MAPK/ERK1/2 signaling and decreased mitosis related proteins might be the underlying mechanism of cell cycle arrest and thus multinucleation. In summary, we confirmed the hypothesis that aberrant cytokinesis and cell fusion resulted in multinucleation in HepG2 cells after SiNPs exposure. Since cell fusion and multinucleation were involved in genetic instability and tumor development, this study suggests the potential ability of SiNPs to induce cellular genetic instability. These findings raise concerns with regard to human health hazards and environmental risks with SiNPs exposure.

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in vivo and in vitro.9−11 SiNPs were able to penetrate cells and enter the cell nucleus, binding to macromolecules including protein and DNA.12,13 It could affect nuclear integrity by forming intranuclear protein aggregates that can cause inhibition of replication and transcription.13 Previously, we have demonstrated that the SiNPs could induce DNA damage, cell cycle arrest and multinucleation in HUVECs, L-02, and HepG2 cells,14−16 suggesting certain genotoxicity of the SiNPs. The existing results of genotoxicity studies were consistent with the fact that the SiNPs were genotoxic.17,18 The genotoxic effects of multinucleation, micronuclei, and chromosomal aberrations contributed to genetic instability and even tumor initiation.19,20 Titanium dioxide nanoparticles have been demonstrated to induce multinucleation, chromosomal instability, and cell transformation in vitro,21 further leading to DNA damage and genetic instability in vivo in mice.22 A recent carcinogenicity study reported that SiNPs could induce 9.4%

INTRODUCTION Silica nanoparticles (SiNPs) are materials intentionally produced, manufactured, or engineered. It is among the most utilized nanomaterials in nanotechnology products.1 SiNPs are industrially used in cosmetics, dentistry, and food ingredients, and in biomedical fields such as gene therapy, medical imaging, and drug delivery.2,3 It has been reported that about 20% of toothpastes contain SiNPs.4 Recently, the silica based diagnostic nanoparticles in the form of “C-dots” (Cornell dots) were approved by Food and Drug Administration (FDA) for stage I human clinical trials.5 The high-volume production of SiNPs and their widespread use might lead to significant environmental, occupational, and consumer exposure. Growing concerns about the safety of SiNPs were raised. International Agency for Research on Cancer (IARC) had classified amorphous silica in group 3 (inadequate evidence for carcinogenicity).6 The Organization of Economic Cooperation and Development (OECD) also listed the SiNPs in the priority of nanomaterials requiring urgent evaluation. Various environmental and toxicological studies have been conducted to investigate the toxic potential of the SiNPs. Most of these studies were described in recent review articles.7,8 Simultaneously, we have also evaluated the safety of SiNPs both © 2015 American Chemical Society

Special Issue: Chemical Toxicology in China Received: November 20, 2014 Published: January 27, 2015 490

DOI: 10.1021/tx500473h Chem. Res. Toxicol. 2015, 28, 490−500

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Chemical Research in Toxicology

were sterilized by an autoclave and dispersed by a sonicator (160 W, 20 kHz, 5 min) to minimize their aggregation before addition into the culture medium. Cells maintained in DMEM without SiNPs were used as the control group. Cellular Uptake of the SiNPs. About 5 × l05 HepG2 cells were seeded in glass bottomed cell culture dishes and cultured in DMEM as described above. After 24 h of cell attachment, the cells were treated with ruthenium(II) hydrate (Ru(phen)32+) interior-labeled SiNPs (62 nm, 50 μg/mL), which were synthesized by a modified Stöber method, at 37 °C in serum-free medium. These red SiNPs had a fluorescent core with a silica shell and no leakage of the dyes from the particles, ensuring the consistency of properties with the nude SiNPs. The detailed characterizations were previously performed and demonstrated the stability of the labeled SiNPs.36 Cells were then washed three times with PBS and fixed with 4% paraformaldehyde at room temperature for 10 min. The cells were washed with 0.1% Triton X100 three times and incubated with Phalloidin-FITC Actin-Tracker Green (Jiancheng, China) at room temperature for 30 min. ActinTracker was dissolved in the mixture of 0.1% Triton X-100 and 3% bovine serum albumin (BSA) (Sigma, USA) for staining the actin filamentous skeleton. After that, the nucleus was stained with 5 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) (Sigma, USA) in PBS for 5 min. Cellular uptake was observed by laser scanning confocal microscopy (LSCM) (Leica TCS SP5, Germany). Transmission Electron Microscope (TEM) Observation. The HepG2 cells were incubated for 24 h with SiNPs (50 μg/mL) and washed with PBS and trypsinized. After centrifugation at 1,500 rpm for 10 min, the cell pellets were fixed in a 0.1 M PBS solution containing 2.5% glutaraldehyde for 3 h. They were then washed with 0.1 M PBS, embedded in 2% agarosegel, postfixed in 4% osmium tetroxide solution for 1 h, washed with distilled water, stained with 0.5% uranyl acetate for 1 h, dehydrated in a graded series of ethanol (30%, 60%, 70%, 90%, and 100%), and embedded in epoxy resin. The resin was polymerized at 60 °C for 48 h. Ultrathin sections obtained with a ultramicrotome were stained with 5% aqueous uranyl acetate and 2% aqueous lead citrate, air-dried, and imaged under a transmission electron microscope (TEM) (JEOL JEM2100, Japan). Multinucleation Assay. Cellular multinucleation was detected by a Giemsa staining kit (KeyGen, China). After exposure to different concentrations of SiNPs for 24 h, the cells were washed with PBS once and then stained by Giemsa dye according to the manufacturer’s instructions. Multinucleated cells and cellular morphological changes were observed using an optical microscope (Olympus IX81, Japan). Cell Cycle Arrest. The cell cycle was measured using a cell cycle detection kit (KeyGen, China). The HepG2 cells were exposed to various concentrations of SiNPs for 24 h, washed with PBS three times, and trypsinized. After centrifugaton at 1,200 rpm for 5 min, the cells were washed once in PBS and fixed in 70% ethanol for at least 24 h. The fixed cells were washed twice with PBS and treated with 100 μL of RNase A at 37 °C for 30 min. Finally, the cells were stained with 400 μL of propidium iodide and incubated in the dark for 30 min. A total of at least 1 × 104 cells for each sample was analyzed by flow cytometry (Becton Dickinson, USA). Immunofluorescence. CPC localization was performed by immunofluorescence. After SiNP exposure for 24 h, the HepG2 cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min. To permeabilize, the fixed cells were incubated with 0.2% TritonX-100 in PBS for 10 min and washed three times in PBS. The fixed and permeabilized cells were blocked with 1% bovine serum albumin in PBS for 1 h and further incubated overnight with the antibody of Aurora B (Invitrogen, USA). The cells were washed in PBS, followed by incubation with Alexa Fluor-555 secondary antibody (Invitrogen, USA) for 1 h at room temperature. Nuclei were stained with 5 μg/mL 4′,6-diamidino-2-phenylindole (DAPI, Sigma) in PBS for 5 min. CPC localization was visualized by a LSCM. Western Blot. Briefly after exposure to varying concentrations of SiNPs for 24 h, HepG2 cells were washed with ice-cold PBS and lysed in ice-cold RIPA lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF) (DingGuo, China) and phosphatase inhibitor for 30 min. After centrifuging the lysates at 12,000 rpm, 4 °C for 10 min, total

tumor incidence in the lungs of female Wistar rats after intratracheal instillation.23 Multinucleated cells are eukaryotic cells that have two or more nuclei within one cytoplasm. They can be divided into syncytium and plasmodium.24 The syncytium is generated by cell fusion and naturally occurs in specialized cells, such as osteoclasts and skeletal muscle cells.25,26 The plasmodium could result from abnormal cytokinesis,27 spindle assembly check-point (SAC) defects,28 or acytokinetic cell division.29 It can be observed in hepatocytes and some tumor cells. Moreover, the defective DNA repair mechanisms could also cause DNA damage-induced multinucleation.30 Multinucleated cells induced by SiNPs was first observed in our previous study,15 and the same case was also reported in other nanoparticles.31−33 Although the multinucleation effects occurred after different nanoparticles exposure, the underlying mechanism of multinucleated cells formation is still unclear. Therefore, it is necessary to investigate the possible ways and potential biological consequences of multinucleated cells resulted from SiNP exposure. The present study was a continuous mechanistic research of the formation of multinucleated cells in HepG2 cells exposed to the SiNPs. Cellular internalization and multinucleation were first investigated. Time-lapse confocal imaging was performed to determine whether the multinucleated cells resulted from cell fusion or abnormal cell division. In a cell mitosis study, the cell cycle and chromosomal passenger complex (CPC) were evaluated. For further mechanism study, cell cycle control proteins in G1/S and G2/M checkpoints along with the MAPK/ERK1/2 signaling pathway were determined.

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MATERIALS AND METHODS

Synthesis of the SiNPs. SiNPs were synthesized using the Stöber method as described before.34 Tetraethylorthosilicate (TEOS) (2.5 mL) was added to a premixed ethanol solution (50 mL) containing ammonia (2 mL) and water (1 mL). The reaction system was kept at 40 °C for 12 h with continuous stirring (150 r/min). After that, the mixture was centrifuged (12,000 r/min, 15 min) to isolate particles. The resulting particles were then washed three times with deionized water and dispersed in 50 mL of deionized water. Characterization of the SiNPs. To measure the average particle size and morphology, SiNPs were suspended in distilled water, sonicated for 5 min, and dropped over the copper grid. The grids were dried and examined under a transmission electron microscope (TEM) (JEOL JEM2100, Japan). A total of 500 particles were randomly chosen and measured by ImageJ software to calculate particle size distribution. The hydrodynamic sizes and zeta potential of SiNPs in distilled water and serum-free DMEM were examined by Zetasizer (Malvern Nano-ZS90, Britain). The SiNP suspensions were dispersed by a sonicator (160 W, 20 kHz, 5 min) (Bioruptor UDC-200, Belgium) to minimize their aggregation before adding into the detecting system. The purity of SiNPs was assessed by ICP-AES (Thermo Fisher Scientific, Switzerland) according to the procedure of element measurement described before.35 Endotoxin in SiNP suspensions was previously detected by the gel clot limulus amebocyte lysate (LAL) assay, and the detection limit was less than 0.125 EU/ mL.9 Cell Culture and Exposure of SiNPs. The human hepatocellular carcinoma HepG2 cells were purchased from Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, USA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified incubator with 5% CO2. For experiments, the cells were seeded in culture plates and treated with SiNPs of certain concentrations (25, 50, 75, and 100 μg/mL). Suspensions of SiNPs 491


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Figure 1. Characterization of the SiNPs. (A) The particles appeared spherical and well dispersed. (B) The hydrodynamic sizes and (C) zeta potential of the SiNPs in both distilled water and serum-free DMEM. (Scale bar: 100 nm).

Figure 2. Cellular uptake and transmission electron microscope (TEM) observation. (A) The control cells and (a) magnification. (B) The cells treated with Ru(phen)32+-labeled SiNPs (red) and (b) magnification. The cell skeleton was stained with Phalloidin-FITC (green) and the cell nucleus with DAPI (blue). (C) The control cells. (D) SiNP internalization and binucleated cells. (E) Magnification of the selected area and electrondense SiNPs dispersed in cytoplasm. (F) SiNP localization and nuclear injury of fragmentation (arrow) and micronucleus (arrow). Time-Lapse Imaging. HepG2 cells were grown on 35 mm glassbottomed dishes for 24 h. Then the nuclei were stained with 1 μg/mL Hoechst 33342 (Sigma, USA) in DMEM for 30 min. After the cells were treated with SiNPs, the dish was placed in a microincubation chamber on an UltraVIEW VoX confocal imaging system (PerkinElmer, USA), which was maintained at 37 °C and equipped with a 5% CO2 supply. Time-lapse observation was performed automatically at multiple locations on the coverslips by collecting fluorescence and differential interference contrast images every 10 min. The series of photographs were then converted into WMV videos using the internal software of Velocity 6.0 (PerkinElmer, USA). Statistical Analysis. Data were expressed as the mean ± SD, and significance was determined by using one-way analysis of variance

cellular protein extracts were determined by performing the bicinchoninic acid (BCA) protein assay (Pierce, USA). Equal amounts of proteins (40 μg) were loaded onto 12% SDS−PAGE and electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After blocking with 5% nonfat milk in Tris-buffered saline (TBS) containing 0.05% Tween-20 (TBST) for 1 h, the membrane was incubated with primary antibodies [1:1000, rabbit antibodies, Cell Signaling Technology (CST), USA] overnight at 4 °C. The membrane was then washed with TBST and incubated with a secondary antibody (CST, USA) for 1 h at room temperature. After washing three times with TBST, the antibody-bound proteins were detected using the ECL chemiluminescence reagent (Pierce, USA). 492


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Figure 3. Representative multinucleation images in which HepG2 cells were incubated with varying concentrations of SiNPs for 24 h. The arrows indicate binucleated and multinucleated cells. (ANOVA) followed by the least significant difference (LSD) test to compare the differences between groups. Differences were considered significant at p < 0.05.

the control group were with normal morphology and round stained nuclei, with only a few multinucleated cells. After the HepG2 cells were treated with SiNPs for 24 h, cellular morphological changes became more obvious. There were also cell number reduction, cellular shrinkage, and chromatin condensation. In addition, with the concentrations of SiNPs increasing, giant cells containing more than one nucleus were frequently observed, suggesting that cell multinucleation increased in a concentration-dependent manner. Real-Time Observation of Multinucleated Cell Formation. To determine whether the SiNP-induced multinucleation was from cell fusion or abnormal cell division, timelapse confocal imaging was performed. The series of photographs and converted videos confirmed that three patterns contributed to the SiNP-induced multinucleation: cell fusion (Figure 4A and Video S1, Supporting Information), karyokinesis without cytokinesis (the nucleus divided in two, while the cytoplasm did not divide during cell division) (Figure 4B and Video S2, Supporting Information), cytokinesis followed by fusion (both the nucleus and cytoplasm divided in two, followed by fusion) (Figure 4C and Video S3, Supporting Information). Localization of the CPC. The CPC is dynamic and localized and plays important roles in different cell division phases. The CPC consists of Aurora B kinase, inner centromere protein (INCENP), Survivin, and Borealin. To investigate the CPC alteration in HepG2 cells after SiNPs exposure, we localized Aurora B kinase (the core of the CPC) and determined the CPC protein expression. In control cells, the fluorescence and punctate patterns of Aurora B were obvious in midbody in anaphase and telophase, respectively (Figure 5A). In contrast, the SiNP-exposed cells only showed diffuse distribution of Aurora B and exhibited binucleate cells without cell division. Moreover, the expression of Aurora B and INCENP were decreased in a dose-dependent manner (Figure

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RESULTS Characterization of SiNPs. SiNP characterizations are summarized in Figure 1A. The particles in TEM images showed spherical shape with an average diameter of 62.2 ± 7.8 nm after 500 particles were randomly chosen and measured. The hydrodynamic sizes and zeta potential were measured by dynamic light scattering (DLS). As depicted in Figure 1B and C, the hydrodynamic sizes of SiNPs were approximately 106.9 ± 0.9 nm and 114.8 ± 1.2 nm with zeta potentials of −36.1 ± 1.2 mV and −43.6 ± 1.6 mV, respectively, in distilled water and serum-free DMEM. Moreover, in serum-containing medium, the hydrodynamic sizes of SiNPs was determined and reported as 187.68 ± 10.75 nm.34 These results showed relatively favorable dispersibility of the SiNPs; in addition, the purity of SiNPs was higher than 99.9%, and no detectable endotoxin was found in the SiNP suspensions.9 Cellular Internalization of SiNPs. Cellular uptake and localization are directly linked to cytotoxicity, and we verified the uptake of Ru(phen)32+-labeled SiNPs (62 nm) by laser scanning confocal microscopy (LSCM). The LSCM images showed that the fluochrome-labeled SiNPs were highly aggregated in cell cytoplasm (Figure 2B). The TEM observation provided further evidence of SiNP uptake. The cells showed internalization of electron-dense SiNPs into intracytoplasmic vacuoles or endosomes (Figure 2D). The particles could also be found randomly in the cytoplasm. Higher magnification insets of the selected areas showed loose aggregates containing multiple nanoparticles, in some instances with retention of the characteristic nanoparticle sizes and shapes (Figure 2E). Cellular Multinucleation. The results of morphological changes and cell multinucleation are shown in Figure 3. Cells in 493


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Figure 4. Photographs extracted from the real-time observation of multinucleated cell formation induced by SiNPs. (A) Cell fusion (arrows). (B) Karyokinesis without cytokinesis (arrows). (C) Karyokinesis without cytokinesis (arrowhead) and cytokinesis followed by fusion (arrows).

and CDK4. As shown in Figure 6B, the increased GSK-3β and decreased Cyclin D1 and CDK4 suggested the delay of G1/S phase transition. Cdc25C could regulate cyclin B1/Cdc2, resulting in the arrest of the G2/M phase. The evaluation of G2/M checkpoint regulators was exhibited in Figure 6C, the expression of Cdc25C, Cdc2, and cyclin B1 were markedly decreased in HepG2 cells after SiNP exposure. MAPK/ERK1/2 Signaling Activation. Cell cycle arrest could be regulated by the extracellular signal-regulated kinases (ERK) in response to DNA damage, which is a subfamily of the mitogen-activated protein kinase (MAPK). We monitored MEK1/2, ERK1/2, and their phosphorylation to determine whether MAPK signaling is associated with cell cycle arrest. As shown in Figure 7, both MEK1/2 and ERK1/2 along with their phosphorylation were obviously increased after HepG2 cells were exposed to SiNPs for 24 h, indicating that the MAPK

5B), suggesting that the SiNPs could induce CPC deficiency through inhibition of CPC component proteins in HepG2 cells. Cell Cycle Arrest of HepG2 Cells Induced by SiNPs. Cell cycle progression of HepG2 cells was analyzed by flow cytometry. As shown in Figure 6A and Table 1, after the HepG2 cells were exposed to SiNPs for 24 h, the percentage of cells in G2/M phase increased gradually in a dose-dependent manner, accompanied by an irregular decline in G0/G1 and S phases. These data revealed that the SiNPs induced G2/M phase arrest in HepG2 cells. Cell Cycle Checkpoint Analysis. Cell cycle arrest occurs in response to DNA damage. To better understand cell cycle arrest and abnormal mitosis, regulators of G1/S and G2/M checkpoint were monitored by assessing corresponding protein expression. Glycogen synthesis kinase-3β (GSK-3β) was involved in G1/S phase transition by inhibiting Cyclin D1 494


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Figure 5. Chromosomal passenger complex (CPC) localization and CPC protein expression. (A) Aurora B kinase is the core of the CPC; in control cells, the punctate Aurora B (red) was obvious in the spindle midzone (arrowhead) and midbody (arrow) in anaphase and telophase, respectively. In contrast, the SiNP-exposed cells showed diffuse fluorescence distribution and exhibited binucleate cells (arrows). (B) All of the CPC component proteins except Survivin were decreased in a dose-dependent manner.

the CPC deficiency further lead to karyokinesis without cytokinesis. The time-lapse confocal imaging visualized our hypothesis that aberrant cytokinesis and cell fusion resulted in multinucleation in HepG2 cells after SiNPs exposure. In our recent publication, we had determined that the cytotoxicity of SiNPs on HepG2 cells was in a dose-dependent manner.11 This may be correlated with the amount of SiNPs taken by HepG2 cells since more SiNPs localizing inside cells could initiate more cytotoxicity.38 Both the laser scanning confocal microscopy images (Figure 2B) and TEM observation provided evidence of SiNPs uptake (Figure 2E). The SiNPs were internalized into HepG2 cells through the endocytic pathway,34 depositing in mitochondria, accumulating in vesicles of endosomes and lysosomes,11 and even entering the nucleus.12 The penetration of silicon carbide based NPs (SiC NPs) inside the cell nucleus was associated with cell division,39 while the uptake of nanoparticles by cells is also influenced by cell cycle phase (G2/M > S > G0/G1).40 Nanoparticles that are

signaling pathway might be responsible for the cell cycle arrest in G1/S and G2/M phase transitions.

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DISCUSSION SiNPs have been widely used in many fields; thus, human exposure is likely to happen during SiNP production, storage, transportation, and consumer use of SiNP-based products. Extensive SiNP exposure brings growing concerns of their potential adverse effects on humans.7,8 This study is an extension of our previous study on the safety evaluation for SiNPs, which previously reported multinucleation induced by SiNPs.15 In the present study, we focused on the significant issue, how the multinucleated cells are generated and further investigated the underlying mechanism in human hepatocellular carcinoma HepG2 cells, which were routinely used to assess the SiNP toxicity.37 We demonstrated for the first time that the SiNPs could induce G1/S and G2/M cell cycle arrest and that 495


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Figure 6. Cell cycle analysis and protein expression at G1/S and G2/M checkpoints. (A) Flow cytometry analysis of the cell cycle. (B) Protein alteration involved in G1/S phase transition. (C) The G2/M checkpoint regulators were decreased in a dose-dependent way.

membrane injury, oxidative stress, DNA damage, and cell death.14,15 Multinucleation induced by SiNPs was previously reported in our studies as adverse effects in response to SiNPsinduced cytotoxicity.15,16 In this study, the number of multinucleated cells was increased in a dose-dependent manner after SiNPs exposure (Figure 3). Multinucleated cells are cells containing more than one nucleus within one cytoplasm. On the basis of the different formation mechanisms, cellular multinucleation could result from cell fusion and karyokinesis without cytokinesis.24 As exhibited in the dynamic observation results (Figure 4), three patterns contributed to the SiNP-induced multinucleation: cell fusion (Figure 4A and Video S1, Supporting Information), karyokinesis without cytokinesis (Figure 4B and Video S2, Supporting Information), and cytokinesis followed by fusion (Figure 4C and Video S3, Supporting Information). Although

Table 1. Cell Cycle Arrest of HepG2 Cells Induced by SiNPsa distribution of cell cycle groups

G0/G1

C 25 50 75 100

70.52 ± 1.35 61.7 ± 1.26* 54.9 ± 0.87* 54.84 ± 2.35* 55.15 ± 0.59*

S 21.07 27.08 27.40 23.62 20.63

± ± ± ± ±

G2/M 0.80 0.46* 0.89* 1.57* 3.03*

8.41 ± 0.81 11.22 ± 1.24* 17.70 ± 0.18* 21.53 ± 0.85* 24.22 ± 2.61*

Data are expressed as the means ± SD from three independent experiments (*p < 0.05).

a

internalized by cells are hard exported from cells. With the amount of intracellular SiNPs increasing, the cells were no longer able to handle these particles, thus causing cytotoxicity,

Figure 7. MAPK/ERK1/2 signaling pathway activation. Both MEK1/2 and ERK1/2 along with their phosphorylation were obviously increased after HepG2 cells were exposed to SiNPs for 24 h. 496


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Figure 8. Schematic representation of dynamic visualized patterns of multinucleation induced by SiNPs. (A) Karyokinesis without cytokinesis. (B) Cytokinesis followed by fusion. (C) Cell fusion.

abnormal cytokinesis and multinucleation in L-02 cells (data not shown). Thus, the deregulated cytoskeleton organization might account for the karyokinesis without cytokinesis in HepG2 cells exposed to SiNPs. Cytokinesis begins with the assembly and constriction of an equatorial contractile ring and ends with the split into two daughter cells. These mitotic processes are associated with the CPC, which is composed of Aurora B kinase, inner centromere protein (INCENP), Survivin, and Borealin.57,58 The CPC is dynamic and localized during different cell division phases and regulates key mitotic events.58,59 In anaphase, the CPC leaves the centromere and relocates to the spindle midzone, while it concentrates at the midbody in telophase.59 The midbody organizes the narrow bridge between the newborn cells and drives the abscission to generate two distinct daughter cells.51 Both the CPC deficiency (Figure 5A) and low expression of the CPC component proteins (Figure 5B) suggest the dysfunction of the cellular constriction and abscission in anaphase and telophase during cytoplasmic division. Consistent with our results, the PEGcoated CdSe QDs were also reported to localize near the contractile ring during cytokinesis and then block contractile ring disassembly.55 These biological processes might contribute to the cytokinesis followed by fusion and thus multinucleation formation. The three visualized dynamic pathways of multinucleation are summarized as a schematic diagram in Figure 8. Besides cytokinesis, DNA damage-induced cell cycle arrest potentially also causes the formation of multinucleated cells since correct timing of the transitions between cell cycle phases is critical for proper cell division. Previously, we have demonstrated that SiNPs could induce DNA damage and G2/M cell cycle arrest in HUVECs and HepG2 cells.14,15 Cell cycle progression is controlled by several cell cycle checkpoints, including G1/S, S, and G2/M.60 These different checkpoints operated cell cycle arrest for completing DNA replication or repair in response to DNA damage. If cell cycle arrest is

cell fusion naturally occurs in specialized cells, such as osteoclasts and skeletal muscle cells,26,41 we observed this phenomenon in HepG2 cells exposed to SiNPs. Cell−cell fusion is a highly regulated and dynamic cellular event based on the merging and rebuilding of cell membranes.42 Functionally, cell−cell fusion in mammals is essential for fertilization and for the formation of both skeletal muscle and the placenta.26 In biotechnology and biomedicine, however, cell fusion has successfully been used for hybrid preparation to generate monoclonal antibodies.43 In addition, this technique has also facilitated the fusing of dendritic cells (DCs) with tumor cells for cancer immunotherapy.44,45 Recently, modified gold nanoparticles were confirmed to bind cells together and lead cell fusion effectively.46 However, in another study, quantum dots (QDs) were not reported to increase the degree of cell− cell fusion.47 For cellular fusion, cells must attach to each other and bring their membranes into close contact. Our group and other researchers have confirmed that SiNPs and other nanoparticles could induce changes on the fluidity, permeability, and structure of cell membrane.34,48−50 During this process, SiNPs might act as the connecting bridge between cells and facilitate cell membrane fusion. With regard to aberrant cytokinesis, karyokinesis without cytokinesis and cytokinesis followed by fusion induced multinucleation in the present study (Figure 4B and C). Cytokinesis is the final step in cell division involving rearrangement of the cellular cytoskeleton and partition of genomic material into two daughter cells.51,52 This process involves cellular cytoskeleton organization, and nanomaterials have been reported to colocalize with the tubulin cytoskeleton,53 causing cytoskeletal disruption and multipolar spindles in different cell lines.21,54,55 However, for polystyrene nanoparticles, they were not observed to affect the cytoskeleton reassembly in both normal and cancer cells.56 In our recent research, cytoskeleton dysfunction was demonstrated to cause 497


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adverse effects of multinucleation, raising the urgent need to define the appropriate conditions for the safe use of SiNPs.

accomplished, the DNA repair machinery can become effective; otherwise, permanent cell cycle arrest at the respective checkpoints might contribute to final DNA damage and genome instability.61 Therefore, we evaluated cell cycle control proteins at the G1/S and G2/M checkpoints to investigate the cell cycle arrest and phases transition in HepG2 cells exposed to SiNPs. As shown in Figure 6A, although the SiNPs induced G2/M phase arrest along with irregular G0/G1 and S phase alteration, two regulars for G1/S checkpoint, Cyclin D1 and CDK4, were decreased in a dose-dependent manner (Figure 6B). Given that both Cyclin D1 and CDK4 control the G1/S phase transition, their decreased expression suggested the transition arrest from G1 to S phase in HepG2 cells. The prolonged G0/G1 phase and decreased expression level of cyclin D and cyclin E were also reported in amino-modified polystyrene (NH2-PS) nanoparticles.56 Moreover, glycogen synthesis kinase-3β (GSK-3β) was reported to inhibit Cyclin D1 to cause G1/S arrest;62 thus, the increased GSK-3β might contribute to the downregulation of Cyclin D1 and CDK4 (Figure 6B). Further investigation of the G2/M arrest showed that Cdc25C, Cdc2, and cyclin B1 were significantly suppressed in HepG2 cells after exposure to SiNPs for 24 h (Figure 6C). Cdc25C downregulated cyclinB1/Cdc2, which is required for G2/M transition of the cell cycle.63 The inhibition of the cyclin B1/Cdc2 complex would result in direct G2/M arrest.64 Both G1/S and G2/M cell cycle arrests were in response to DNA damage for DNA repair to maintain genome stability. The ERK1/2 (extracellular signal-regulated kinases), subfamilies of the mitogen-activated protein kinase (MAPK) family, plays critical roles in cell cycle progression and DNA damage response.65,66 These kinases were regulated by upstream kinases, MEK1/2. As shown in Figure 7, the phosphorylation of both MEK1/2 and ERK1/2 was increased in SiNP-treated HepG2 cells, suggesting the activation of the MAPK signaling pathway. Therefore, the activated ERK1/2 signaling pathway might play positive roles in the SiNP-induced cell cycle arrest and ultimate multinucleation. Both cell−cell fusion and aberrant cytokinesis account for multinucleation formation in HepG2 cells exposed to SiNPs. Cell−cell fusion and multinucleation have been considered to be involved in genetic instability and tumor development. Cell fusion is discussed as a mechanism for the transmission of malignancy from tumor to normal host stromal cells.67,68 In addition, multinucleation is an important characteristic in tumor progression and correlates to tumor grade.69 Although the IARC has classified the carcinogenicity of amorphous silica particles in group 3, the silica particles have been shown to elicit tumors in experimental animals.23,70 As reported, after repeated instillation of SiNPs, a statistically significant tumor response (9.4%) was observed in Wistar rats.23 In this study, multinucleation and abnormal mitosis were observed, suggesting that SiNPs might have the ability to induce cellular genetic instability or tumor initiation. In the present study, we demonstrated that cell fusion, karyokinesis without cytokinesis, and cytokinesis followed by fusion contributed to the multinucleated cell formation in HepG2 cells after SiNP exposure. The aberrant cytokinesis and ultimate multinucleation might result from CPC deficiency and cell cycle arrest in G1/S and G2/M phases, which is involved in the potential mechanism of MAPK/ERK1/2 signaling activation and mitosis related protein downregulation. This study clearly identified and characterized the SiNP-induced

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ASSOCIATED CONTENT

* Supporting Information S

Real-time confocal imaging of multinucleated cell formation; Video S1, cell fusion; Video S2, karyokinesis without cytokinesis; Video S3, both karyokinesis without cytokinesis and cytokinesis followed by fusion. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86 010 83911507. Fax: +86 010 83911507. E-mail: [email protected]. Funding

This work was supported by the National Natural Science Foundation of China (No. 81172704). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Professor Wensheng Yang from Jilin University for the preparation of silica nanoparticles.

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ABBREVIATIONS SiNPs, silica nanoparticles; DAPI, 4′,6-diamidino-2-phenylindole; CPC, chromosomal passenger complex; GSK-3β, glycogen syntheses kinase-3β; DLS, dynamic light scattering; TEM, transmission electron microscope; ICP-AES, inductively coupled plasma atomic emission spectrometry; LSCM, laser scanning confocal microscopy

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REFERENCES

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