Abstract
Background. Despite the rapid development of nanotechnology, the biological significance of TiO2 nanoparticles (NPs), possibly released from dental materials, is not well-understood. We investigated the effect of TiO2 NPs on the sensitivity of human oral squamous cell carcinoma (OSCC) cell line (HSC-2) to five popular chemotherapeutic agents. Materials and Methods. Viable cell number was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The aggregation and cellular uptake of TiO2 NPs were assessed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), respectively. Adsorption of TiO2 NPs to anticancer drugs was assessed by the antitumor activity recovered from the TiO2 NP-free supernatant. Results: When mixed with culture medium, TiO2 NPs instantly aggregated, and some particles were incorporated into the cells, exclusively in the vacuoles. TiO2 NPs showed no cytotoxicity nor hormetic growth stimulation at lower concentrations. Doxorubicin, melphalan, 5-fluorouracil and gefitinib were cytotoxic, whereas docetaxel was cytostatic with or without TiO2 NPs. TiO2 NPs, at wide concentration ranges (0.2-3.2 mM), did not significantly affect the adsorption of NPs to any of these anticancer drugs, nor affected their cytotoxic or cytostatic activity. Conclusion: This experimental study demonstrated for the first time that TiO2 NP do not affect the antitumor potential of chemotherapeutic agents against the HSC-2 OSCC cell line.
Nanotechnology is the manipulation of materials with at least one dimension sized from 1 to 100 nanometers on an atomic and molecular scale, especially for fabrication of macroscale products. Public expenditure on nanotechnology has dramatically risen during the past 10 years (1). Nanoparticles (NPs) have a greater surface-to-volume ratio (per unit mass) than non-nanoscale particles composed of the same material, and therefore are more reactive, as predicted from the laws of quantum physics (2). Thus, nanotechnology holds promise for a broad variety of new biological, biomedical and biochemical applications. The small size of NPs allows their incorporation into cells via endocytosis (3-6) and they, therefore, may affect the cellular function (7). Metal oxide NPs are used as the components of sunscreens, making it necessary to perform the risk assessment of NPs and their secondary effects (8).
Titanium dioxide (TiO2) is very insoluble and thermally stable. This oxide is used as a white pigment in ceramics, cosmetics and medicines. However, the safety of TiO2 NPs is controversial. Some reports describe the safety of inhaled or ingested particles (9), while others reported their biohazardous effects, based on the fact of increasing the risk of asthma susceptibility in the offspring when pregnant mice were exposed to respirable TiO2 NPs (10).
Nanomaterials have a high tendency to aggregate in cell culture medium, and this property may cause significant variation in the derived biological data, depending on the culture conditions (11). Furthermore, the magnitudes of their modulatory effects may vary considerable among different types of NP. For example, Mn2O3 NPs inhibited O2 consumption by 50% at 170 mg/l, and damaged cell membrane by approximately 30% at 1,000 mg/l, while TiO2, ZrO2 and Fe2O3 NPs had little or no toxicity against the yeast, Saccharomyces cerevisiae (11). Similarly, the antitumor potential of TiO2 NPs is also controversial. TiO2 NPs reduced the viable cell number of osteosarcoma U-2OS and chondrosarcoma SW1353 cell lines dose-dependently (12) and induced apoptosis of the human lung cancer A549 cell line (13) by its pro-oxidant action. On the other hand, TiO2 NPs had no apparent cytotoxicity against human kidney glomerular mesangial IP 15 and epithelial proximal HK-2 cell lines, even at high doses (14). Furthermore, how TiO2 NPs affect the sensitivity of oral cells against dental treatment is not clear.
We investigated whether TiO2 NPs (i) become aggregated in culture medium; (ii) are incorporated into cells and show cytotoxicity; (iii) adsorb to five popular antitumor agents [doxorubicin (anthracycline antibiotic), melphalan (alkylating agent), 5-fluorouracil (5-FU, pyrimidine analog), docetaxel (taxane), gefitinib (protein tyrosine kinase inhibitor)]; and (iv) modify antitumor action of these agents, in the human oral squamous cell carcinoma (OSCC) cell line HSC-2.
Materials and Methods
Materials. The following chemicals and reagents were obtained from the indicated companies: Dulbecco's modified Eagle's medium (DMEM), GIBCO BRL (Grand Island, NY, USA); fetal bovine serum (FBS), JRH Bioscience (Lenexa, KS, USA); TiO2 NPs (nanopowder, anatase phase, particle size <25 nm, purity 99.7% trace metal basis, MW 79.87 g/mol) and melphalan Aldrich (St. Louis, MO, USA); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), culture plastic dishes, 6-well and 96-mircowell plates, Becton Dickinson (Franklin Lakes, NJ, USA); doxorubicin and dimethyl sulfoxide (DMSO) Wako Pure Chem Ind. (Osaka, Japan); 5-FU, Kyowa (Tokyo, Japan); docetaxel, Toronto Research Chemicals (Ontario, Canada); and gefitinib, LC Laboratories®, PKC Pharmaceuticals, Inc. (Woburn, MA, USA).
Assay for TiO2 NP topography. TiO2 NPs were vortexed and suspended by sonication in distilled water with a bath-type sonicator (Tokyo Cho-onpa Giken Co. Tokyo, Japan) for 1 min at room temperature, since water was found to be the best vehicle to produce a homogeneous suspension of TiO2 NPs most efficiently, as compared with alcohol, phosphate-buffered saline without calcium and magnesium [PBS(−)] or medium (data not shown). A drop of TiO2 NPs was set on an aluminum stub, dried for 48 h at room temperature, and covered with 50 nm of gold sputtering. The topographical surface was then observed with scanning electron microscope (SEM) (JSM-6360LV; JEOL, Tachikawa, Japan) with secondary electrons at ×10,000 magnification by 15 kV.
Cell culture. HSC-2 cells (Riken Cell Bank, Tsukuba, Japan) were cultured at 37°C in DMEM supplemented with 10% heat-inactivated FBS, 100 units/ml, penicillin G and 100 μg/ml streptomycin sulfate under a humidified atmosphere with 5% CO2. Cells were then harvested by treatment with 0.25% trypsin-0.025% EDTA-2Na in PBS−(−) and either subcultured or used for experiments.
Assay for cytotoxic activity. Cells (2×104 cells/0.1 ml) were inoculated into each well of 96-microwell plates and incubated for 48 h to achieve the complete cell adherence. The medium was replaced with 0.1 ml of fresh medium containing 0, 0.2, 0.4, 0.8, 1.6 and 3.2 mM of TiO2 NPs. After 30 min, the cells were treated with different concentrations of doxorubicin, melphalan, 5-FU, docetaxel or gefitinib and incubated further for 48 h. The relative viable cell number was then determined by the MTT method. In brief, the culture medium was replaced with MTT (0.2 mg/ml), and cells were incubated for 4 h at 37°C. After removal of medium, the formazan product was dissolved with DMSO, and the absorbance at 540 nm of the lysate, which reflects the mitochondrial activity, was determined by using a microplate reader (Multiskan; Biochromatic Labsystem, Osaka, Japan) (15). The 50% cytotoxic concentration (CC50) was determined from the dose–response curve and the mean CC50 (±S.D.) value of each anticancer drug was calculated from three independent experiments each of which were performed in triplicate.
Detection of aggregation of TiO2 nanoparticles (NPs) by scanning electron microscope (SEM). Micrograph was taken with secondary electrons at ×10,000 magnification by 15kV.
Interaction of TiO2 NPs with anticancer drugs. Doxorubicin (final: 1 mM), melphalan (10 mM), 5-FU (10 mM), docetaxel (0.1 mM) or gefitinib (10 mM) was mixed with TiO2 NPs (final: 8 mM) in a total volume of 40 μl of DMSO, and stood at room temperature for 10 min. The mixture was centrifuged at 21,880 ×g for 5 min, and the supernatant, free from TiO2 NPs, was collected. Near-confluent HSC-2 cells were incubated for 48 h with serially diluted supernatant, and the relative viable cell number was then determined by MTT method as described above and compared with that of treatment without the use of TiO2 NPs.
Intracellular uptake of TiO2 NPs. Cells were treated with 0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6 and 3.2 mM of TiO2 NPs for 3 h. The cells were then washed three times with cold PBS(−) and fixed for 1 h with 2% gultaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at 4°C, scraped with a rubber policemen, dehydrated and then embedded in Araldite M (Ciba–Geigy Swiss; NISSHIN EN Co., Ltd., Tokyo, Japan). Thin sections were stained with uranyl acetate and lead citrate, and were then observed under a JEM-1210 transmission electron microscope (TEM) (JEOL) (magnification: ×5,000) at an accelerating voltage of 100 kV (16).
Intracellular uptake of TiO2 nanoparticles (NPs) demonstrated by a transmission electron microscope (TEM). Near-confluent HSC-2 cells were incubated for 3 h with 0 (A), 0.05 (B), 0.1 (C), 0.2 (D), 0.4 (E), 0.8 (F), 1.6 (G) and 3.2 (H) mM of TiO2 NPs. The cells were then washed three times with cold PBS(−) and fixed for 1 h with 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at 4°C for the observation of the fine cell structure by TEM.
Statistical analysis. Data are expressed as the mean±standard deviation (S.D.). Statistical analysis was carried out with Kruskal–Wallis test and multiple comparisons by Mann–Whitney test with SPSS (Statistical Package for the Social Sciences, Chicago, IL, USA). Differences were considered significant at p<0.05.
Results
Topography and intracellular uptake of TiO2 NPs. We prepared a homogeneous TiO2 NP suspension by sonication in water. However, TiO2 NPs easily aggregate to make clusters during the preparation for SEM (Figure 1) or when added to culture medium (as demonstrated by TEM) (Figure 2). This may be due to the interactions between the amine and carboxylic groups on the cell membrane (negative charge) and the surface of TiO2 NPs (positive charge) (11). Interaction or attraction between the cells and TiO2 NPs may produce high biocompatibility. Some TiO2 NPs were incorporated into the cells, specifically in the vacuoles, in a dose-dependent fashion (Figure 2).
Possible interaction of TiO2 NPs and anticancer drugs. High affinity of cells for TiO2 NPs prompted us to investigate whether TiO2 NPs show similar affinity toward test compounds. To test this possibility, we first incubated TiO2 NPs with each anticancer drug (doxorubicin, melphalan, 5-FU, docetaxel, or gefitinib) in DMSO, and then collected the supernatant that did not contain TiO2 NPs. If a significant preparation of TiO2 NPs were adsorbed to the anticancer drugs, the concentration of anticancer drugs recovered from the supernatant would be reduced. The results showed this was not the case, with superimposable dose–response curves, regardless of the presence or absence of TiO2 NPs (Figure 3). These results show adsorption of TiO2 NPs to anticancer drugs to be low or having little effect on bioavailability of these drugs.
Interaction between anticancer drugs and TiO2 nanoparticles (NPs). A: Flow chart of experimental procedure. B: The five anticancer drugs were first mixed with (closed symbols) or without (open symbols) TiO2 NPs (64 mM), and the TiO2-free supernatant was collected after centrifugation at 21,880 ×g for 5 min. Near-confluent HSC-2 cells were then incubated for 48 h with serially-diluted supernatant (0, 0.0078, 0.0156, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1, 2%) to determine the viable cell number by the 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The highest concentration (2%) of supernatant without pre-incubation with TiO2 NPs contained 10 μM doxorubicin, 100 μM melphalan, 100 μM 5-FU, 1 μM docetaxel and 100 μM gefitinib, respectively. Each value represents the mean±S.D. of triplicate assays.
Effect of TiO2 nanoparticles (NPs) on growth-inhibitory activity of anticancer drugs against HSC-2 cells. HSC-2 cells were first pre-treated for 30 min in the presence of 0, 0.2, 0.4, 0.8, 1.6 or 3.2 mM TiO2 NPs, and then incubated for a further 48 h with doxorubicin (A, F, K), melphalan (B, G, L), 5-fluorouracil (5-FU) (C, H, M), docetaxel (D, I, N) or gefitinib (E, J, O) to determine the viable cell number by the 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The data of first (A-E), second (F-J) and third (K-O) experiments are shown. Each value represents the mean±S.D. of triplicate assays. Absorbance value of control cells treated with 0, 0.2, 0.4, 0.8, 1.6 or 3.2 mM TiO2 NPs without anticancer drug was 2.060±0.358 (100%), 1.915±0.332 (93%), 2.039±0.479 (99%), 1.957±0.432 (95%), 2.120±0.399 (103%) and 2.027±0.367 (98%), respectively (n=45, triplicate × three times experiments × five anticancer drugs). There was statistically no difference between 0 and other concentrations of TiO2 NPs (p>0.30). It should be noted that concentration ranges for some compounds were changed in experiments 1 to 3, so as to determine the CC50 values.
Effect of TiO2 nanoparticles on the antitumor potential of five chemotherapeutics drugs against human oral squamous cell carcinoma cell line HSC-2. Values represent the mean±S.D. of three independent experiments.
Effect on cytotoxic activity of anticancer drugs. Next, we investigated whether TiO2 NPs modify the cytotoxicity of anticancer drugs. HSC-2 cells were first pre-incubated for 30 min with TiO2 NPs (0, 0.2, 0.4, 0.8, 1.6 or 3.2 mM), and then added with increasing concentrations of each anticancer drug and cells incubated for a further 48 h to determine the viable cell number. We found that TiO2 NPs had no cytotoxicity nor hormetic growth stimulation at lower concentrations (Figure 4). Doxorubicin, melphalan, 5-FU, and gefitinib were cytotoxic, completely killing the cells at higher concentrations, whereas docetaxel was cytostatic, regardless of the presence or absence of TiO2 NPs (Figure 4). The dose-dependent curves of their antitumor activities were superimposable, regardless of the concentration of TiO2 NPs (Figure 4). Repeated experiments confirmed that the CC50 values at any TiO2 NP concentrations were not significantly (p>0.05) different from those of the controls (without TiO2 NPs), maintaining the same order of antitumor potency: docetaxel > doxorubicin > melphalan > gefitinib, 5-FU (Table I). This demonstrates that TiO2 NPs did not potentiate or reduce the antitumor potential of the five anticancer drugs tested.
Discussion
Nanoparticles have a wide applications in the field of medicine and dentistry due to their antibacterial, antifungal and antiviral potential (17-19). However, the study of the antitumor potential of TiO2 NPs has been limited. Recently, it has been reported that TiO2 NPs alone showed little or no toxicity against rat glioma C6, RG2, mouse melanoma B16 and human glioma U373 cell lines, and copper–TiO2 NP complex were significantly less cytotoxic than copper alone, suggesting some protective effect of the complex formation with TiO2 NPs, although its cytotoxicity was slightly higher than that of cisplatin (20). The Copper–TiO2 complex maybe incorporated into mitochondria to reduce ATP synthesis and formation of nitrogenous bases, and reach the cell nucleus to create links with DNA base pairs by interaction or groove binding and finally induce apoptotic cell death (20). It has been also reported that TiO2 NPs doped with Au and Pt effectively killed cells of the human erythroleukemia tumor cell line K562 (21).
The uptake mechanisms for TiO2 NPs are still unclear, however, it has been reported that TiO2 NPs were taken-up and accumulated in the vacuoles, endosomes and lysosomes, or localized in the cytoplasm possibly due to lysosomal membrane rupture (20, 22), confirming the present finding with HSC-2 OSCC cell line that the TiO2 NPs were sequestered only into the vacuoles, where interestingly they became aggregated again (Figure 2). It was recently reported that TiO2 NPs were transported through plasma membrane via transmembrane toll-like receptor (TLR 4) in human hepatocellular carcinoma HepG2 and human chronic myelogenous leukemia K562 cell lines (23).
The present study demonstrated that TiO2 NPs, at a wide range of concentrations (up to 3.2 mM), did not adsorb to any of the five anticancer drugs (doxorubicin, melphalan, 5-FU, docetaxel, gefitinib), nor affected their growth–inhibitory activity against the HSC-2 against human OSCC cell line, supporting a previous report with other types of cultured cells (18). The present study demonstrated, to our knowledge, for the first time, that TiO2 NPs themselves exhibit no cytotoxicity nor hormetic growth stimulation at low concentrations, quite differently than many toxic substances, environmental hormones, inorganic compounds, and even irradiation, modulate the growth of cultured cells in a bi-phasic fashion, stimulating or inhibiting the growth of cultured cells at lower and higher concentrations, respectively, as seen in previous studies (24).
TiO2 NPs slightly reduced the cytostatic activity of docetaxel (Table I), however, the CC50 values varied considerably due to the cytostatic (not cytotoxic) property of this compound. Taken together with the previous reports, we conclude that TiO2 NPs showed no cytotoxicity nor hormetic growth stimulation at lower concentrations, nor affected the antitumor potential of chemotherapeutic agents when used against of OSCC cell line HSC-2. However, this would not mean that TiO2 NPs are absolutely safe substances in the oral environment, since TiO2 NPs aggravated inflammation induced by interleukin-1β in human gingival fibroblast (unpublished data). The interaction between OSCC and inflammatory human gingival fibroblast cells in the oral cavity remains to be investigated.
- Received November 11, 2013.
- Revision received November 15, 2013.
- Accepted November 18, 2013.
- Copyright © 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
References
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