Cytotoxic effects and the mechanism of three types of magnetic nanoparticles on human hepatoma BEL-7402 cells
© Kai et al; licensee Springer. 2011
Received: 7 April 2011
Accepted: 29 July 2011
Published: 29 July 2011
The evaluation of the toxicity of magnetic nanoparticles (MNPs) has attracted much attention in recent years. The current study aimed to investigate the cytotoxic effects of Fe3O4, oleic acid-coated Fe3O4 (OA-Fe3O4), and carbon-coated Fe (C-Fe) nanoparticles on human hepatoma BEL-7402 cells and the mechanisms. WST-1 assay demonstrated that the cytotoxicity of three types of MNPs was in a dose-dependent manner. G1 (Fe3O4 and OA-Fe3O4) phase and G2 (C-Fe) phase cell arrests and apoptosis induced by MNPs were detected by flow cytometry analysis. The increase in apoptosis was accompanied with the Bax over-expression, mitochondrial membrane potential decrease, and the release of cytochrome C from mitochondria into cytosol. Moreover, apoptosis was further confirmed by morphological and biochemical hallmarks, such as swollen mitochondria with lysing cristae and caspase-3 activation. Our results revealed that certain concentrations of the three types of MNPs affect BEL-7402 cells viability via cell arrest and inducing apoptosis, and the MNPs-induced apoptosis is mediated through the mitochondrial-dependent pathway. The influence potency of MNPs observed in all experiments would be: C-Fe > Fe3O4 > OA-Fe3O4.
Over the past few decades, as nanotechnology and materials science has progressed incredibly swiftly, nanomaterials have been widely applied in many fields including medicine, pharmaceuticals, manufacturing technologies, electronics, and telecommunications [1–3]. In particular, the surge of interest in nanomaterials has significantly expanded the breadth of research on magnetic nanoparticles (MNPs) during the recent decade. Due to their multifunctional properties, MNPs are explored for various biomedical applications such as contrast agents for MRI [4, 5], targeted drug and gene delivery [6, 7], cell sorting , hyperthermia , or combinations of multiple applications, both diagnostic and therapeutic . Some MNPs, such as bowel contrast agents (Lumiren® and Gastromark®) and liver/spleen imaging (Endorem® and Feridex IV®) [11, 12], are already in the market. Moreover, the potential applications of MNPs (e.g., bare Fe3O4 and C-Fe) have expanded into other fields including environmental restoration [13, 14] and agriculture [15–18]. Some researches indicate that MNPs would accumulate in aquatic organisms , crops  for further entry into the food chain. Humans are therefore increasingly exposed to various kinds of MNPs, directly or indirectly.
Along with the expanding applications of MNPs, the potential toxic effects of MNPs have been of wide concern [20–23]. Multiple results show that MNPs significantly reduce cell viability of human macrophage, epithelial cell lines , human mesothelioma , and inhibit the normal formation of PC12 neuronal cell morphology . At higher concentrations, DMSA-coated MNPs decrease mitochondrial activity of human fibroblasts . Meanwhile, the cytotoxicity of MNPs is found in a dose-dependent manner .
Nevertheless, the cytotoxicity data of MNPs is difficult to compare since the toxic effects of MNPs are influenced by many parameters such as size distribution, surface coating, magnetic properties, etc. . Numerous studies can be found that, quite often, report on seemingly contradicting findings since different cell types will interact with the same particle in different ways . Therefore, it is crucial to choose the cell line for the cytotoxicity assessment of specific MNPs. Several pharmacokinetic reports indicate that liver is the most important organ involving the bioaccumulation and clearance procedures of MNPs [29–31]. Furthermore, the cytotoxicity studies of MNPs are limited by the fact that cytology mechanism remained unexplored.
In the present study, human hepatoma BEL-7402 cell line was selected as the model specimen for cytotoxicity assessment, and the aims were to evaluate the cytotoxicity of Fe3O4, OA-Fe3O4, and C-Fe and to elucidate the mechanisms of their cytotoxicities. MNPs internalization was observed by transmission electron microscopy (TEM) and cell viability was determined by tetrazolium salt-based (WST-1) assay. For the study of the mechanism of cytotoxicity, cell cycle and apoptosis were analyzed by flow cytometry. To further elucidate the apoptosis pathway, the mitochondrial membrane potential (MMP), the Bax and cytochrome C protein expression, and caspase-3 activity were investigated.
Results and discussion
MNPs uptake by human hepatoma BEL-7402 cells
The dose-dependent cytotoxicity of nanoparticles
MNPs influence on the cell cycle
MNPs affected cell cycle distribution of BEL-7402 cells
Cell cycle (%)
Cells with reversibly damaged DNA will accumulate in G1, S, or G2/M phase , while cells that carry irreversibly damaged DNA will undergo apoptosis [37, 38]. Hence, it is necessary to further analyze the cell apoptosis to fully interpret the toxic effects of MNPs on BEL-7402 cells.
MNPs-induced apoptosis of BEL-7402 cells
This apoptosis result is consistent with cytotoxicity trends shown in WST-1 assay. The mechanisms of cytotoxic effects of MNPs on BEL-7402 cells may be implemented through cell cycle arrest and inducing apoptosis.
Assay of mitochondria-dependent apoptosis in BEL-7402 cells after MNPs
Apoptosis is a tightly controlled process in which cell death is executed through the activation of specific signaling pathways [41, 42]. Although it is well established that many organelles contribute to apoptosis, extensive research shows that nanoparticles induced cell apoptosis via mitochondria-dependent pathway [43, 44]. As an indicative of mitochondria involvement in the apoptosis, the apparently swollen mitochondria with lysing cristae were observed by TEM (Figure 1). Therefore, we speculate that BEL-7402 cell apoptosis was induced by MNPs through mitochondria-dependent pathway.
To sum up, our results indeed suggested that all three types of MNPs can induce apoptosis in BEL-7402 cells through mitochondria-dependent pathway. Moreover, the influence potency of MNPs on the mitochondria-dependent apoptosis would be: C-Fe > Fe3O4 > OA-Fe3O4, and all in a dose-dependent manner.
In this paper, cytotoxic effects and the mechanism of Fe3O4, OA- Fe3O4, and C-Fe MNPs on BEL-7402 cells were studied. A dose-dependent cytotoxicity pattern was found in all three types of MNPs via WST-1 assay. The results of flow cytometric analysis revealed that the cytotoxicity of MNPs is implemented through cell cycle arrest and inducing apoptosis. The results of mitochondrial membrane potential, Western blots for Bax and cytochrome C, and caspase-3 activation further elucidate that MNPs induce apoptosis through mitochondria-dependent pathway. Moreover, the influence potency of MNPs observed in all experiments would be: C-Fe > Fe3O4 > OA-Fe3O4.
Recent studies show that the cytotoxicities of many MNPs could be due to reactive oxygen species (ROS) induction [55, 56]. And accompanied with the MNPs degradation, the altered cellular iron pool can then affect cellular functionality by altering the level of transferrin receptor expression and can affect cellular proliferation capacity by altering the expression of cyclins and cyclin-dependent kinases in cell cycle [57, 58]. Therefore, the metabolism, ROS determination and transferrin receptor expression will be the next step for further reveal of the cytotoxicities of Fe3O4, OA- Fe3O4, and C-Fe.
Materials and methods
RPMI-1640 and fetal bovine serum were purchased from Gibco, Invitrogen Corp., Carlsbad, CA, USA. PI and RNase I were obtained from Sigma, St. Louis, MO, USA. Alexa Fluor® 488 annexin V/Dead Cell Apoptosis Kit was obtained from Invitrogen, USA. The primary antibodies to Bax, cytochrome C, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The goat anti-Mouse IgG-HRP, mouse anti-rabbit IgG-HRP, and Potent ECL kit were purchased from Multisciences, Hanzhou, China. Caspase-3/CPP32 Colorimetric Assay Kit and Mitochondria/Cytosol Fractionation Kit were purchased from BioVision, Mountain View, CA, USA. Total Protein Extraction Kit and BCA Protein Assay Kit were obtained from Applygen Technologies Inc., Beijing, China. The lipophilic cationic dye JC-1 (5, 5, 6, 6-tetrachloro-1, 1, 3, 3-tetraethylbenzimidazol-carbocyanine iodide) was obtained from ChemoMetec, Allerød, Denmark. WST-1 Cell Proliferation and Cytotoxicity Assay Kit was purchased from Beyotime Institute of Biotechnology, Haimen, China. All other reagents are analytical or cultured grade purity.
Cell culture and preparation of MNPs
Human hepatoma BEL-7402 cell line was a gift kindly provided by Medical College of Xiamen University (Xiamen, China). The cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum. Incubation was carried out at 37°C in a humidified 5% CO2 incubator. For all experiments, the cells were in the exponential growth phase. The MNPs used in this study were: (1) Fe3O4 MNPs, purchased from Aladdin (Shanghai, China); (2) OA-Fe3O4 MNPs, purchased from Jinke (Maanshan, China); (3) C-Fe MNPs, purchased from Junye (Shenzhen, China). The purity of three types MNPs are 99.9% and the size distribution of particles are 10-30 nm. Nanoparticle stock suspensions (10 mg/mL) were prepared by UV-sterilization and dispersing a known weight of nanoparticles in RPMI-1640 medium under ultrasonication. The stock suspensions were sonicated for 20 min to distribute the particles, and then dilutions were made in complete media to achieve desired testing concentrations. The test suspensions were sonicated for 20 min before use. Untreated controls were exposed to complete media only, and processed identical to the exposed cells.
Cells (2 × 106) were seeded into 100-cm2 petri dishes. Cells were allowed to attach for 24 h and were then treated with each MNPs test suspensions for 24 h in a concentration of 0.5 mg/mL. Then, the cells were collected and fixed with 2.5% glutaraldehyde buffered in 0.1 M PBS overnight at 4°C. The samples were washed with PBS, and post-fixed in 1% osmium tetroxide at 4°C for 1 h. After dehydration in series concentrations of ethanol and infiltration in acetone, cells were embedded in Epon 812, and ultra-thin sections cut with glass knives were stained with uranyl acetate and lead citrate, and viewed under JEM 2100 TEM (JEOL, Tokyo, Japan).
To determine cell toxicity/viability, BEL-7402 cells (0.5 × 104, 100 μL) were plated onto 96-multiwell plates (Costar, Corning, NY, USA) and incubated for 24 h. Then, cells were exposed to various concentrations (0.01-2 mg/mL) of each MNPs test suspensions for 24 h. Afterwards, the old media was discarded and replaced with 100 μL of new complete media. WST-1 solution (10 μL) was added to each well, followed by incubation for 2.5 h. Absorbance at 490 nm (reference at 630 nm) was measured by a spectrophotometric microplate reader (Bio-tek ELX800, BioTek Instruments, Winooski, VT1, USA). A negative control was provided using the culture medium without the nanoparticles. Each of the particle concentrations and the controls was seeded in eight wells. The percentage cell viability was calculated in term of absorbency in cells treated with MNPs relative to that in cells exposed to culture media alone.
Cell cycle assay
A cell cycle assay was carried out by staining the DNA with PI and analyzing the fluorescence using flow cytometry. Following exposure of the BEL-7402 cells to each MNPs for 24 h, any damaged cells can detach from the plate and become suspended in the medium, necessitating medium storage. Briefly, the cells were harvested, washed with PBS, and fixed in ice-cold 70% of ethanol at -20°C before use. After resuspension, cells were washed, and incubated with 100 μL PI (400 μg/mL) and 100 μL RNase I (1 mg/mL) at 37°C for 15 min. Cells were analyzed with an EPICS XL flow cytometer (Beckman Coulter Inc., Fullerton, CA, USA) and the data were consequently evaluated by Mod-Fit (Verity Software, Topsham, ME, USA).
Detection of apoptosis by annexin V assay
Apoptosis was evaluated using Alexa Fluor® 488 Annexin V/PI Apoptosis Kit. BEL-7402 cells were treated with two concentrations (0.05 and 1 mg/mL) of each MNPs for 24 h. After exposure, the cells (5-10 × 104) were harvested, washed and resuspended with PBS. The Annexin V/PI staining of cells followed the manufacturer's instructions. Then the samples were analyzed with EPICS XL flow cytometer (Beckman Coulter, USA). The results were expressed as the number of apoptotic cells per thousand cells counted.
The mitochondrial membrane potential was measured by flow cytometry using JC-1 dye. JC-1 changes its fluorescence from green at 535 nm (monomer state) to orange at 590 nm (aggregate state) as it enters in mitochondria of intact cells. When the mitochondrial membrane potential is affected, JC-1 returns to its green monomeric state. All procedures were carried out according to the manufacturer's instructions. The cell treatments were the same with "Detection of apoptosis by annexin V assay" section. In brief, approximately 2 × 106 cells were harvested, washed, resuspended in PBS (1 mL) and stained with 12.5 μL JC-1(200 μg/mL) for 15 min at 37°C in the dark. Both fluorescences emitted by the cells were monitored by flow cytometry and the ratio orange/green fluorescence was calculated. The data was determined by analyzing 10,000 cells using an EPICS XL flow cytometer (Beckman Coulter, Fullerton, CA, USA), and Cell Quest software (Becton Dickinson, San Jose, CA, USA).
Western blot analysis of Bax and cytochrome C
The cell treatments were the same with "Detection of apoptosis by annexin V assay" section. Approximately 1 × 107 cells per sample were harvested. The protein samples of Bax and cytochrome C were extracted using Total Protein Extraction Kit and Mitochondria/Cytosol Fractionation Kit, respectively. Protein contents were quantified using the BCA protein assay kit and stored at -70°C. Protein (20 μg per lane) was resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membranes (PVDF, Millipore Corporation, Billerica, MA, USA). The transblotted membrane was washed, blocked, and incubated at 4°C overnight with anti-Bax antibody and anti-cytochrome C antibody, respectively. Immunodetection with the secondary HRP-conjugated antibody and chemiluminescence using Potent ECL Kit were performed according to the manufacturer's protocol. Equal protein loading was verified by probing with anti-β actin antibody. Densitometric analysis for the blots was performed with Bandscan image software.
Caspase-3 activity assay
The activity of caspase-3 was determined using caspase-3/CPP32 Colorimetric Assay Kit. The cell treatments were the same with "Detection of apoptosis by annexin V assay" section. All procedures were carried out according to the manufacturer's instructions. Briefly, 2 × 107 BEL-7402 cells were lysed by the solution provided in the assay kit and the protein concentration was measured using BCA protein assay kit. For caspase-3 activity assay, equal amounts of total cell lysates were mixed with a caspase-specific substrate DEVD-pNA in a 96-well plate in triplicate. After incubation at 37°C for 2 h, the caspase-3-mediated cleavage of DEVD-pNA into free pNA was measured using spectrophotometric microplate reader (Bio-tek ELX800, USA) at 405 nm. The results were expressed as absorbance compared with control.
All results were expressed as mean values ± S.D. Statistical analysis was performed according to the Student's t test. The probability values of P < 0.05 were considered as significant.
This work was financially supported by the Major Research plan of the National Natural Science Foundation of China (Grant No. 90923042), National Key Technologies R & D Program of China (Grant No. 2007BAD07B05).
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