Induction of Cell Death in Mesothelioma Cells by Magnetite

Article pubs.acs.org/journal/abseba

Induction of Cell Death in Mesothelioma Cells by Magnetite Nanoparticles Shofu Matsuda,† Airi Hitsuji,† Takuya Nakanishi,† Hong Zhang,† Akane Tanaka,‡ Hiroshi Matsuda,‡ and Tetsuya Osaka*,† †

Graduate School of Advanced Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan Division of Animal Life Science, Institute of Agriculture, Tokyo University of Agriculture and Technology, 3-8-1 Harumi-cho, Fuchu, Tokyo 183-0057, Japan

‡

S Supporting Information *

ABSTRACT: Nanoparticle uptake and cell death following addition of magnetite nanoparticles (MNPs) with a diameter of ∼10 nm were evaluated in three histological types of human mesothelioma cells, NCI-H28 (epithelioid), NCI-H2052 (sarcomatoid), and MSTO211H (biphasic) cells, and human breast cancer MCF-7 cells. Dosedependent cell death was observed in MSTO-211H cells but not in MCF-7 cells, although cellular uptake of MNPs was observed in both cell types. Mesothelioma NCI-H28 and NCI-H2052 cells showed behavior more similar to that of breast cancer MCF-7 cells than that of mesothelioma MSTO-211H cells. DNA fragmentation and microarray analyses suggested that MNPs induced transforming growth factor β2 related apoptosis in MSTO-211H cells. On the other hand, the viability of human mesothelioma cells containing MNPs with a diameter of ∼40 nm was investigated after exposure to an alternating magnetic field. Temperature increase under the alternating magnetic field and high rates of cell death were observed in all three histological types of human mesothelioma. KEYWORDS: mesothelioma, magnetite nanoparticles, cytotoxicity, uptake

1. INTRODUCTION Malignant pleural or peritoneal mesothelioma is an aggressive tumor that develops after asbestos exposure,1−4 for which the primary treatment is surgical resection,2,3,5 radiation therapy,2,3 and chemotherapy.2,3,6 However, survival rates are low because mesothelioma is usually situated deep in the body and is resistant to chemotherapeutic agents. Moreover, surgical removal is invasive, and chemotherapy has negative side effects. Although multimodal therapy combining two or more treatments is proposed to improve the survival rate and quality of life,2,7−9 the most effective combination is still contested. Therefore, more effective treatments for mesothelioma, for prolonging survival or effecting a cure, are strongly required. Nanoparticles of iron oxides such as magnetite, Fe3O4, are being intensively investigated for applications in the medical field and have been developed in recent years as contrast agents in magnetic resonance imaging (MRI), carriers in drug delivery systems, and heating elements for magnetic hyperthermia.10−16 We previously demonstrated the synthesis of magnetite nanoparticles (MNPs) with the size tuned in the range of 10−40 nm in mean diameter,17 examined their uptake as well as their effect on cell viability in human breast cancer MCF-7 cells,18,19 human umbilical vein endothelial cells,18 and mouse embryonic stem cells,20 and suggested the safety of the © XXXX American Chemical Society

synthesized MNPs as well as their potential for use in hyperthermia. In this study, we investigated nanoparticle uptake and cell death induced by the addition of MNPs to human mesothelioma cells and considered the future applications of MNPs to mesothelioma treatment. It should be noted that the influence of nanomaterials on cells, for example, cytotoxicity, is often cell-type-specific.21−25 Brunner et al. compared the in vitro cytotoxicity of oxide nanoparticles, including iron oxide (Fe2O3 but not Fe3O4), in human mesothelioma MSTO-211H with that in rodent 3T3 fibroblast cells and reported that MSTO-211H cells were highly sensitive to Fe2O3, while 3T3 cells were not greatly affected.21 All three major histologic subtypes of mesothelioma (i.e., epithelioid, sarcomatoid, and biphasic) were investigated in this study. To the best of our knowledge, the work reported here represents the first investigation of the cellular uptake and cytotoxicity of MNPs by three histological cell types, in addition to their cell death induced by MNPs subjected to an alternating magnetic field. Received: January 7, 2015 Accepted: June 23, 2015

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DOI: 10.1021/acsbiomaterials.5b00009 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

UltraStructure Research Institute Co., Ltd. (Aichi, Japan) with a JEOL JEM1200EX at an acceleration voltage of 80 kV. To prepare specimens for TEM observation, cells were washed with 0.1 M phosphate buffer, fixed with 2% osmium tetroxide, dehydrated with 50−100% ethanol, embedded in EPON812, and finally sectioned using an ultramicrotome. The amount of MNPs incorporated into a NCI-H28, NCI-H2052, or MSTO-211H cell was evaluated using a previously described procedure.19,20 HCl, trichloroacetic acid solution, H2O2, and potassium thiocyanate solution were used to dissolve cells with MNPs, to precipitate the cellular proteins, to oxidize Fe2+ into Fe3+, and to form a thiocyanate−iron(III) complex; finally, the absorbance at 480 nm was measured using a JASCO V-550 spectrophotometer (JASCO International Co. Ltd., Tokyo, Japan). 2.4. Detection of DNA Fragmentation in Apoptotic Cells. MSTO-211H cells were cultured at a density of 5 × 105 per well in the presence of 800 μg of ∼10 nm MNPs as described above. After 24 h of incubation, a DNA fragmentation assay was performed using a quick apoptotic DNA ladder detection kit (Invitrogen) according to the manufacturer’s protocol. After extraction of chromosomal DNA, DNA fragmentation was analyzed by agarose gel electrophoresis. The DNA fragments were viewed under UV light and photographed. 2.5. Microarray Analysis. NCI-H28, NCI-H2052, and MSTO211H cells were cultured for 24 h at a density of 5 × 105 cells per well in the presence of 800 μg of MNPs as described above. Total RNA isolation and gene expression microarrays were performed at Cell Innovator Inc. (Fukuoka, Japan). Total RNA was isolated from the cerebellum of individual animals using TRIzol reagent (Invitrogen) and purified using the SV total RNA isolation system (Promega) according to the manufacturer’s instructions. A microarray analysis of gene expression was performed using an Agilent SurePrint G3 human gene expression microarray 8×60K v2. The cRNA was amplified and labeled using Agilent’s low input quick amp labeling kit and hybridized to a 60K Agilent 60-mer oligomicroarray according to the manufacturer’s instructions. All hybridized microarray slides were scanned with an Agilent scanner. Relative hybridization intensities and background hybridization values were calculated using the Agilent Feature extraction software (9.5.1.1). Microarray data were normalized using the quantile algorithm. 2.6. Evaluation of Cell Death with MNPs Subjected to Alternating Magnetic Field. MNPs with a diameter of ∼40 nm, such as ferromagnetic MNPs, were employed here. NCI-H28, NCIH2052, MSTO-211H, and MCF-7 cells were cultured for 24 h with 800 μg of MNPs, and then the cellular uptake and cytotoxicity of ∼40 nm MNPs were evaluated. Prior to application of the AC magnetic field, we collected 5 × 105 cells containing MNPs by magnetic separation. The cells were exposed to an AC magnetic field with an output power of 4.3 kW and an electric current of 569.1 A at a frequency of 325 kHz for 20 min, using an EASYHEAT inductionheating device (Alonics, Ltd., Tokyo, Japan) equipped with a threeturn coil with an outer diameter of 40 mm. The strength of the magnetic field was calculated to be 536 Oe. During the 20 min exposure to the AC magnetic field, the sample temperature was measured with an Anritsu FL-2000 fiber thermometer (Tokyo, Japan). The percentage of nonviable cells was determined using a flow cytometer as described in section 2.3, immediately after the magnetic field exposure.

2. MATERIALS AND METHODS 2.1. Cell Culture. Human mesothelioma NCI-H28, NCI-H2052, and MSTO-211H cells (American Type Culture Collection) were maintained in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO) containing 10% fetal bovine serum (FBS; ThermoFisher Scientific, Waltham, MA) and 1% penicillin−streptomycin (Sigma-Aldrich). Human breast cancer MCF-7 cells were cultured in Eagle’s minimum essential medium (EMEM; Sigma-Aldrich) with 10% FBS, 5% Lglutamine (Invitrogen, Life Technologies, Grand Island, NY), 1% MEM nonessential amino acid (Gibco, Life Technologies), and 1% antibiotic−antimycotic (Gibco). All cells were incubated at 37 °C under a 5% CO2 atmosphere. 2.2. Synthesis of Magnetite Nanoparticles. MNPs were synthesized by hydrolysis of an aqueous solution containing ferrous chloride (FeCl2·4H2O) and ferric chloride (FeCl3·6H2O), using essentially the same procedure as described in a previous paper,17 except for the use of spermine or N,N′-bis(3-aminopropyl)butane-1,4diamine as a base instead of 1,6-hexanediamine.18,19 Iron salts and spermine were purchased from Kanto Chemical Co. Ltd. (Tokyo, Japan) and Sigma-Aldrich Japan, respectively. According to the synthetic procedure previously reported for ∼10 and ∼40 nm MNPs,19 50 mL of an aqueous solution containing ferrous chloride and ferric chloride at a molar ratio of 2:1 (for ∼10 nm MNPs) or 1:0 (for ∼40 nm MNPs), the total amount of which was 0.05 mol dm−3, was mixed with 50 mL of an aqueous solution containing 0.125 mol dm−3 of spermine, and the mixture was stirred at room temperature. After being stirred for 24 h (for ∼10 nm MNPs) or 4 h (for ∼40 nm MNPs), the black precipitate was separated using a permanent magnet and washed several times with ultrapure water and ethanol. The final products were obtained in the form of a black powder after being dried in a desiccator at room temperature. The shape and size of the products were observed by transmission electron microscopy (TEM) with a JEOL JEM-1011 microscope (Tokyo, Japan) or a Hitachi H7650 microscope (Tokyo, Japan) operated at an acceleration voltage of 100 kV. The crystal structure of the products was characterized by X-ray diffraction (XRD) patterns recorded using a RINT ULTIMA III diffractometer (Rigaku, Tokyo, Japan) with Cu Kα radiation (1.5418 Å). The ζ-potential of the specimens dispersed in water at pH ∼7 was evaluated using an Otsuka Electronics ELS-8000 light-scattering photometer (Osaka, Japan). 2.3. Evaluation of Cytotoxicity and Cellular Uptake. NCIH28, NCI-H2052, MSTO-211H, and MCF-7 cells were cultured in 6well plates at a density of 5 × 105 cells per 3 mL of medium in each well and then incubated without or with ∼10 nm MNPs at 200, 400, 600, and 800 μg per well. After incubation for 24 h, the cells were washed with Dulbecco’s phosphate-buffered saline (DPBS; Gibco) to remove excess MNPs. The morphology of MSTO-211H and MCF-7 cells was observed using a Nikon TE2000-U microscope (Tokyo, Japan). Cell pellets were obtained by centrifugation at 1200 rpm for 5 min and suspended in 1 mL of DPBS supplemented with 6% FBS, followed by staining with 1 μL of 42 μM propidium iodide (PI) in dimethyl sulfoxide and 2 μL of 4.3 mM thiazole orange (TO) in water. TO and PI were purchased from BD Biosciences, Becton, Dickinson and Co. (Franklin Lakes, NJ) as part of the BD cell viability kit. Immediately following this treatment, the cells were analyzed with a BD FACSCanto II flow cytometer. PI stains dead cells only, whereas TO stains both living and dead cells. Dead cells were recognized by their PI-positive response, and cells were distinguished from MNPs by TO-positive staining. Cellular uptake of MNPs was measured using side scatter (SSC) section. The ratio of cells containing MNPs to the total number of cells was determined in the following way. In a flow cytometer, light scattering occurs when a cell deflects incident laser light. The scattered light is collected as forward scatter (FSC) and SSC. The SSC depends on the inner complexity of the cell because SSC measures refracted and reflected light at a side angle to the laser path. Therefore, high values along the SSC axis would indicate that the cells contained MNPs.18,20 To discuss the cellular uptake of MNPs, TEM observation of NCI-H28, NCI-H2052, and MSTO-211H incubated with 800 μg of MNPs was performed at the Hanaichi

3. RESULTS 3.1. Characterization of Magnetite Nanoparticles. Figure 1 shows TEM images, histograms of particle size distribution, and XRD patterns of the MNPs employed in this study. TEM observation showed that particle shape and size were consistent with our previous report.17−20 One hundred nanoparticles were counted to determine their size distribution; the mean particle diameters were 10.3 and 42.7 nm, shown in Figure 1B,D, respectively. The XRD patterns of the samples (Figure 1E) matched the standard pattern of Fe3O4 (JCPDS# 19-629), and the d value of the lattice spacing of (311) was B


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γ-Fe2O3 of 2.518 Å (JCPDS# 39-1346), similar to our previous report.17 The ζ-potentials of ∼10 and ∼40 nm MNPs, measured in water at pH ∼7, were +10.7 and +16.8 mV, respectively, which is attributable to the cationic form of the amine groups in spermine adsorbed on the surface of the nanoparticles as a protecting reagent.18 3.2. Cell Morphology. We observed the morphology of MSTO-211H and MCF-7 cells cultured without and with the addition of ∼10 nm MNPs. Figure 2A shows an optical microscopic image of MSTO-211H cells incubated for 24 h without MNPs. A mixture of spindle-like cells (mainly in Figure 2A) and polygonal cells (partly in Figure 2A), which is characteristic of biphasic mesothelioma MSTO-211H cells, was observed. The spindle-like cells formed multilayers and were in cell−cell contact. In contrast, after 24 h of incubation with 200 μg of MNPs, some of the MSTO-211H cells shrunk to a needle-like shape. MSTO-211H cells treated with MNPs were confirmed to have lower adhesive capacity than the control cells (Figure 2B). In contrast, no morphological differences were observed between MCF-7 cells incubated without (Figure 2C) and with 200 μg of MNPs (Figure 2D). At a high dose (800 μg) of MNPs (Supporting Information Figure S1), more MSTO-211H cells appeared shrunken than at a dose of 200 μg, although the large number of MNPs rendered observation difficult. It should be noted that, prior to the addition of MNPs, the MSTO-211H cells were in satisfactory condition for experiments, based on the results shown in the Supporting Information (Figure S2). 3.3. Cell Death Associated with the Addition of Magnetite Nanoparticles. To consider the influence of MNPs on cells, cell death following addition of MNPs was also investigated. Figure 3A shows the dose dependence of the percentage of nonviable cells, as evaluated by flow cytometry, with the addition of 0, 200, 400, 600, and 800 μg of MNPs to mesothelioma NCI-H28, NCI-H2052, and MSTO-211H cells and breast cancer MCF-7 cells (5 × 105 cells for each treatment). Dose-dependent cell death was observed in MSTO-

Figure 1. Characterization of the MNPs employed in this study. TEM images (A,C) and size distribution (B,D) of ∼10 nm MNPs (A,B) and ∼40 nm MNPs (C,D). XRD patterns with Cu Kα radiation (E) of ∼10 nm MNPs (a) and ∼40 nm MNPs (b).

calculated to be 2.528 and 2.534 Å for (a) and (b), respectively, using the Bragg equation for the peak observed at 2θ ∼ 36°. The calculated values were concordant with the standard value for Fe3O4 of 2.532 Å (JCPDS# 19-629), not with the value for

Figure 2. Cell morphology of MSTO-211H (A,B) and MCF-7 (C,D) cells after incubation for 24 h without (A,C) and with (B,D) MNPs. In B and D, 200 μg of ∼10 nm MNPs was administered to 5 × 105 cells. Scale bar: 50 μm. C


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Figure 3. Dependence of the percentage of nonviable cells (A) and the percentage of cells containing MNPs (B) on the amount of ∼10 nm MNPs added to 5 × 105 NCI-H28 cells (diamonds), NCI-H2052 cells (squares), MSTO-211H cells (circles), and MCF-7 cells (triangles).

Figure 4. TEM images of a NCI-H28 cell (A), a NCI-H2052 cell (B), and a MSTO-211H cell (C) containing MNPs.

211H cells; at a dose of 200 μg, ∼20% nonviable cells were observed, reaching ∼50% at a dose of 800 μg. In contrast, the percentage of nonviable MCF-7 cells remained constant (∼4%) at doses ranging from 0 to 800 μg. It should be noted here that, among the mesothelioma cells, the response of epithelioid NCI-H28 and sarcomatoid NCI-H2052 cells differed from that of biphasic MSTO-211H cells. The percentage of nonviable NCI-H28 cells remained almost constant, as low as ∼10%, at doses ranging from 0 to 800 μg, whereas the percentage of nonviable NCI-H2052 cells slightly but gradually increased with the dose, from ∼5% at a dose of 200 μg to ∼15% at doses of 600 and 800 μg. The dependence of cell death on the incubation time was also investigated in MSTO-211H cells, at a dose of 800 μg MNPs (shown in the Supporting Information, Figure S3). The percentage of nonviable MSTO-211H cells gradually increased with the incubation time, from ∼5% at 2 h to ∼50% at 24 h; the percentage of nonviable control cells, without MNPs, remained below ∼8% with incubation times ranging from 2 to 24 h. 3.4. Cellular Uptake of Magnetite Nanoparticles. Figure 3B shows the ratio of the number of cells containing MNPs to the total number of cells, as evaluated by flow cytometry, under conditions similar to those in Figure 3A. The percentage of cells containing MNPs was shown to increase gradually as the MNP dose increased for both MSTO-211H and MCF-7 cells; it reached ∼70 and ∼90% for MSTO-211H and MCF-7 cells, respectively, at a dose of 800 μg of MNPs. The percentage of NCI-H28 and NCI-H2052 cells containing

MNPs, in contrast, reached ∼80% even at a dose as low as 200 μg. To understand the cellular uptake of MNPs in more detail, we examined the state and amount of MNPs incorporated in a single cell for the three types of mesothelioma cells. TEM images (Figure 4) revealed that (i) MNPs were incorporated into cells as aggregates and (ii) MNPs were not present in the nucleus but were present in the cytoplasmic endoplasmic reticulum in all three types (A, epithelioid NCI-H28; B, sarcomatoid NCI-H2052; and C, biphasic MSTO-211H cells). The images shown in Figure 4 are representative of multiple TEM images not shown here for each cell types. These results are similar to previous observations for human breast cancer MCF-7 cells.18 On the other hand, several differences were observed among the three histological types, for example, in the size of the vesicles (endosomes) containing the MNPs and the density of MNPs in these vesicles. Figure 5 shows the dose dependence of the uptake of MNPs by mesothelioma cells. In all three mesothelioma cell types, the quantity of MNPs incorporated per cell increased linearly with the dose of MNPs; ∼150 and ∼650 pg of MNPs were incorporated per cell at doses of 200 and 800 μg, respectively. 3.5. Apoptotic DNA Fragmentation. Figure 6 shows the results of the DNA fragmentation assay. Following agarose gel electrophoresis, DNA laddering was observed for MSTO-211H cells incubated with MNPs (lane 3), whereas control MSTO211H cells without addition of MNPs did not show either laddering or smearing (lane 2). This result provides evidence D


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Figure 7. Expression level of TGF-β2 in all three histological types of human mesothelioma cells (NCI-H28, NCI-H2052, and MSTO211H) cultured without and with ∼10 nm MNPs. The control signal intensity was set to “1”.

Figure 5. Dependence of the amount of MNPs incorporated per cell on the amount of ∼10 nm MNPs added to 5 × 105 NCI-H28 cells (diamonds), NCI-H2052 cells (squares), and MSTO-211H cells (circles).

MNPs were investigated and compared with those of ∼10 nm MNPs (Table 1). With the addition of ∼40 nm MNPs, the Table 1. Nanoparticle Uptake and Cell Death at a Dose of 800 μg of MNPs ∼40 nm MNPs cell type NCI-H28 NCI-H2052 MSTO211H MCF-7

cell death (%)

a

∼10 nm MNPs

cellular uptake (%)

b

cell deatha (%)

cellular uptakeb (%)

12.0 11.9 30.0

72.5 89.3 64.6

9.6 14.6 47.5

78.2 85.7 65.7

3.1

91.5

4.1

85.7

Percentage of nonviable cells, as evaluated by flow cytometry. b Percentage of cells containing nanoparticles, as evaluated by flow cytometry. a

percentages of nonviable cells and cells containing nanoparticles were similar in tendency to those of ∼10 nm MNPs. It should be noted here that ∼40 nm particles showed a cytotoxic effect on MSTO-211H lower than that on ∼10 nm particles. Figure 8 shows the percentage of nonviable cells, as evaluated by flow cytometry, following 20 min of exposure to an AC magnetic field. It should be noted that this examination was performed on MNP-containing cells collected by magnetic separation. In all three cell types, the percentage of nonviable

Figure 6. DNA fragmentation analyzed by 1.2% agarose gel electrophoresis: the 1 kb DNA ladder marker (lane 1), control MSTO-211H cells without addition of MNPs (lane 2), and MSTO211H cells after 24 h of incubation with ∼10 nm MNPs (lane 3).

for the occurrence of apoptotic DNA fragmentation in MSTO211H cells after incubation with MNPs. 3.6. Gene Expression. To consider why only MSTO-211H cells showed high rates of cell death, we examined differences in gene expression without and with the addition of MNPs in three types of mesothelioma cells. As shown in Figure 7, transforming growth factor-β2 (TGF-β2) was observed to overexpress specifically in MSTO-211H cells; ∼43-fold upregulation of TGF-β2 was observed in MSTO-211H cells treated with MNPs, whereas TGF-β2 expression was unaltered by addition of MNPs in NCI-H28 and NCI-H2052 cells. 3.7. Induction of Cell Death with ∼40 nm Magnetite Nanoparticles after Exposure to an AC Magnetic Field. We employed ∼40 nm MNPs here because ferromagnetic MNPs with a diameter of ∼40 nm were shown to be much more effective for inducing cell death of MCF-7 cells, by their heat generation under an AC magnetic field, than superparamagnetic MNPs with a diameter of ∼10 nm in our previous study.19 First, the cellular uptake and cytotoxicity of ∼40 nm

Figure 8. Percentage of nonviable human mesothelioma cells without MNPs (green bars) and containing MNPs (red bars) after exposure to an AC magnetic field (325 kHz, the strength was calculated to be ∼500 Oe) for 20 min. For each histological type, 800 μg of ∼40 nm MNPs was added to 5 × 105 cells. For details, please see section 2.6. E


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ACS Biomaterials Science & Engineering cells containing MNPs was >80%: ∼84% for NCI-H28, ∼86% for NCI-H2052, and ∼91% for MSTO-211H. During exposure to the AC magnetic field, the increase in temperature of the samples was observed; the average temperature between 400 and 1200 s was ∼54, ∼52, and ∼40 °C for NCI-H28, NCIH2052, and MSTO-211H cells, respectively. In contrast, when the same examination was performed on NCI-H28, NCIH2052, and MSTO-211H cells without MNPs, the percentage of nonviable cells was lower than 5% in all three types because the temperature of the samples did not increase under the AC magnetic field.

Therefore, the overexpression of TGF-β2 caused by the addition of MNPs is suggested to result in the induction of apoptosis specific to MSTO-211H cells. Although the reason for cell-type-specific overexpression of TGF-β2 has not been elucidated, it is interesting to note that Khan et al. reported that uptake of MNPs affects the TGF-β signaling pathway in HeLa cells.28 When ∼40 nm MNPs were employed, a significant degree of cell death was induced not only in MSTO-211H but also in NCI-H28 and NCI-H2052 cells when cells containing MNPs were subjected to an AC magnetic field. The increase in temperature originating from heat generated by the ∼40 nm MNPs could cause the high degree of cell death observed, as also demonstrated in MCF-7 cells in our previous paper,19 although the reason why NCI-H28 and NCI-H2052 cells reached a higher temperature than MSTO-211H cells remains unclear at present. In general, cell death by heating is known to be induced by both apoptosis and necrosis via DNA damage in a temperature-dependent manner.29,30 It should be noted that death of MSTO-211H cells is partially induced by the simple addition of MNPs; this would contribute to our observation that MSTO-211H cells showed the highest percentage of nonviable cells (∼91%) at the lowest temperature (∼40 °C) among the three cell types. The results of the in vitro experiments performed in the present study demonstrated the potential of MNPs for future application to mesothelioma treatment via the following two approaches: (i) the use of the specific apoptotic effect of MNPs on biphasic MSTO-211H cells and (ii) the use of heat generation by MNPs subjected to an AC magnetic field, which induced a high degree of cell mortality in all three major histologic subtypes of mesothelioma cells (epithelioid NCIH28, sarcomatoid NCI-H2052, and biphasic MSTO-211H cells). The ∼10 nm MNPs could be applied to the former approach, whereas the ∼40 nm MNPs show particular promise for the latter.

4. DISCUSSION Because we observed shrinkage of human mesothelioma MSTO-211H cells following the addition of MNPs (Figure 2B), unlike other cell types in our previous studies,18−20 we focused our attention on the specific cytotoxic effect of MNPs on mesothelioma cells. Using flow cytometry, we demonstrated that induction of cell death associated with addition of MNPs was observed in mesothelioma MSTO-211H (biphasic) cells but not in human breast cancer MCF-7 cells. Other histological types of mesothelioma cells, NCI-H28 (epithelioid) and NCIH2052 (sarcomatoid), showed greater similarity to MCF-7 than to MSTO-211H cells. It should be noted that the percentage of cells containing MNPs increased with the MNP dose not only in MSTO-211H but also in MCF-7, NCI-H28, and NCIH2052 cells (Figure 3), and the amount of MNPs incorporated per cell increased linearly with the dose for all three types of mesothelioma cells (Figure 5). Thus, the cytotoxic effect of MNPs appears to be specific to biphasic mesothelioma cells. As mentioned in the Introduction, Brunner et al. investigated the in vitro cytotoxicity of oxide nanoparticles using MTT and Hoechst assays and showed that MSTO-211H cells were highly sensitive to Fe2O3, while 3T3 cells were not greatly affected.21 Although they speculated that the high sensitivity of MSTO compared to 3T3 cells could be attributed to phagocytotic activity of MSTO cells that is higher than that of 3T3 cells, the reasons for this cell-type-specific response remain unclear. Our study is the first to show that MNPs show distinct in vitro cytotoxicity in MSTO-211H (biphasic) cells, among three histologic mesothelioma subtypes. We further analyzed the dose dependence of the cell viability and cellular uptake of MNPs in MSTO-211H cells (shown as circles in Figure 3). At a dose of 200 μg to 5 × 105 of MSTO211H cells, approximately 20% of cells were nonviable and 40% contained MNPs. At a dose of 800 μg, the percentages of nonviable cells and MNP-containing cells reached ∼50 and ∼70%, respectively (Figure 3A,B). Although the percentage of nonviable cells tended to be lower than that of MNPcontaining cells, both percentages gradually increased as the dose increased, suggesting a certain relationship between cell death and cellular uptake of MNPs. Concerning the mode of cell death, the appearance of DNA ladders in agarose gel electrophoresis (Figure 6) is related to the endonuclease activation with subsequent cleavage of DNA into nucleosomal fragments, which characterizes apoptosis. Furthermore, we found that TGF-β2 was overexpressed specifically in MSTO211H cells but not in NCI-H28 or NCI-H2052 cells. TGF-β2 is reported to have physiological activities, including induction of apoptosis, involving generation of reactive oxygen species.26,27 It should be noted that the mode of cellular uptake and the intracellular location and state of incorporated MNPs, observed by TEM (Figure 4), were similar in all three histological types.

5. CONCLUSION We investigated the cellular uptake and cytotoxicity of magnetite nanoparticles to mesothelioma cells. The MNPs employed in this study induced apoptosis in MSTO-211H (biphasic) cells but had little cytotoxic effect on NCI-H28 (epithelioid), NCI-H2052 (sarcomatoid), or breast cancer MCF-7 cells in vitro, although the mode of cellular uptake and the quantity of MNPs incorporated per cell were similar in all cell types investigated here. This biphasic-type specific death of mesothelioma cells was demonstrated for the first time in the present work. Exposure of cells containing MNPs to an AC magnetic field induced a high degree of cell death not only in MSTO-211H but also in NCI-H28 and NCI-H2052 cells. For mesothelioma cells, there have been no reports of the heatinduced cell death with MNPs under an AC magnetic field, to the best of our knowledge. The apoptotic cell death of MSTO211H cells induced by the simple addition of MNPs has a potential to be used in mesothelioma treatment.

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.5b00009. Figures S1−S3 (PDF) F


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Corresponding Author

*Tel: +81-3-5286-3202. Fax: +81-3-3205-2074. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by a Grant-in-Aid for Specially Promoted Research (Grant Number 20002006) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, and by a Waseda University Grant for Special Research Projects (2013B-279 and 2014B249). Also, this work was partly supported by the Center of Innovation Program from Japan Science and Technology Agency (JST). We are grateful to Mr. Shunpei Asuka (APRO Life Science Institute, Inc.) for help with microarray analysis. S.M. acknowledges the Leading Graduate Program in Science and Engineering, Waseda University, from MEXT, Japan.

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Induction of Cell Death in Mesothelioma Cells by Magnetite

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