Akiyoshi Taniguchi

Molecular mechanism of DNA damage induced by titanium dioxide nanoparticles in toll-like receptor 3 or 4 expressing human hepatocarcinoma cell lines

BackgroundTitanium dioxide nanoparticles (TiO2 NPs) are widely used in the biological sciences. The increasing use of TiO2 NPs increases the risk of humans and the environment being exposed to NPs. We previously showed that toll-like receptors (TLRs) play an important role in the interactions between NPs and cells. Our previous results indicated that TLR4 increased the DNA damage response induced by TiO2 NPs, due to enhanced NP uptake into the cytoplasm, whereas TLR3 expression decreased the DNA damage response induced by TiO2 NPs because of NP retention in the endosome. In this study, we explored the molecular mechanism of the DNA damage response induced by TiO2 NPs using TLR3 or TLR4 transfected cells. We examined the effect of TLR3 or TLR4 over-expression on oxidative stress and the effect of DNA damage induced by TiO2 NPs on gene expression levels.ResultsOur results showed evidence for elevated oxidative stress, including the generation of reactive oxygen species (ROS), with increased hydrogen peroxide levels, decreased glutathione peroxidase, and reduced glutathione and activated caspase-3 levels in cells exposed for 48 h to 10 μg/ml TiO2 NPs. These effects were enhanced by TLR4 and reduced by TLR3 over-expression. Seventeen genes related to DNA double-strand breaks and apoptosis were induced, particularly IP6K3 and ATM.ConclusionOur results indicated that TiO2 NPs induced ROS, and the above molecules are implicated in the genotoxicity induced by TiO2 NPs.

Role of toll-like receptors 3, 4 and 7 in cellular uptake and response to titanium dioxide nanoparticles

Abstract Innate immune response is believed to be among the earliest provisional cellular responses, and mediates the interactions between microbes and cells. Toll-like receptors (TLRs) are critical to these interactions. We hypothesize that TLRs also play an important role in interactions between nanoparticles (NPs) and cells, although little information has been reported concerning such an interaction. In this study, we investigated the role of TLR3, TLR4 and TLR7 in cellular uptake of titanium dioxide NP (TiO2 NP) agglomerates and the resulting inflammatory responses to these NPs. Our data indicate that TLR4 is involved in the uptake of TiO2 NPs and promotes the associated inflammatory responses. The data also suggest that TLR3, which has a subcellular location distinct from that of TLR4, inhibits the denaturation of cellular protein caused by TiO2 NPs. In contrast, the unique cellular localization of TLR7 has middle-ground functional roles in cellular response after TiO2 NP exposure. These findings are important for understanding the molecular interaction mechanisms between NPs and cells.

Detection of cellular response to titanium dioxide nanoparticle agglomerates by sensor cells using heat shock protein promoter

Nanotechnology is becoming increasingly important for products used in our daily lives, such as the masses of titanium dioxide nanoparticle agglomerates (TiO2 NPs) used in the pharmaceutical industry, for cosmetic products, or for pigments. Meanwhile, a serious lack of detailed information concerning the interaction between the nanomaterials and cells limits their biological and medical applications. Sensing technology is very important for understanding these interactions. We have shown that TiO2 NPs induce heat shock protein 70B (HSP70B) mRNA [Okuda‐Shimazaki et al., 2010. Int J Mol Sci 11:2383–2392]. In the current work, sensor cells for detection of cellular responses to NPs were prepared by transfecting an HSP70B promoter–reporter plasmid. First, to find suitable cells for detection, five different mammalian cell lines were chosen as potential sensor cells. The results showed TiO2 NP response in some cell lines, although different sensor cells had different TiO2 NP response levels, as heat shock response ability is important for the detection. Then, we studied the TiO2 NP time‐course response and dose response. The results indicated that our sensor cells can detect TiO2 NP cellular responses. Our work should aid in understanding the interactions between bio‐nanomaterials and cells. Biotechnol. Bioeng. 2012; 109: 3112–3118.

A three-dimensional collagen-sponge-based culture system coated with simplified recombinant fibronectin improves the function of a hepatocyte cell line

Hepatocytes are widely used in pharmaceutical drug discovery tests, but their hepatic functions decrease rapidly during in vitro culture. Many culture systems have been devised to address this problem. We here report that a three-dimensional (3D) collagen-based scaffold coated with simplified recombinant fibronectin (FN) enhanced the function of a hepatocyte cell line. The developed culture system uses a honeycomb collagen sponge coated with collagen-binding domain (CBD)-cell attachment site (CAS), a chimeric protein comprising the CBD and CAS of FN. The function of HepG2 cells grown on honeycomb collagen sponge coated with CBD-CAS was investigated by determining the messenger RNA (mRNA) expression levels of several genes. The mRNA expression level of albumin increased 3.25 times in cells grown on CBD-CAS-coated honeycomb collagen sponge for 3xa0days; the expression level of CCAAT/enhancer binding protein (C/EBPα) increased 40-fold after 1xa0d and up to 150-fold after 3xa0d. These results suggested that CBD-CAS-coated honeycomb collagen sponge could improve the functions of hepatocytes by inducing C/EBPα expression. The activation of cytochrome P450 (CYP) enzymes in HepG2 cells grown on CBD-CAS-coated honeycomb collagen sponge was measured at the mRNA level and was found to increase between two and six times compared to cells grown without the CBD-CAS coating, showing that this culture system induced CYP gene expression and thus may be useful in drug metabolism assays.

Induction of albumin expression in HepG2 cells using immobilized simplified recombinant fibronectin protein

Optimization of the extracellular environment is very important for hepatocyte function in vitro. We expressed new chimeric proteins of the collagen-binding domain (CBD) with cell attachment site (CAS) of fibronectin to enhance hepatocyte function, and the CBD-CAS proteins were immobilized on collagen-coated plates. We hypothesized that the high density of CAS would increase activity of the integrin-dependent intracellular signaling pathway, thus inducing hepatocyte function. Expression of albumin in the human hepatocyte cell line HepG2 was assessed on CBD-CAS-immobilized dishes. The results indicated that the CBD-CAS-immobilized plates induced albumin expression. Immobilized CBD-CAS induced activation of focal adhesion kinase and integrin-ligand clustering on the cell membrane. These results suggest that immobilized CBD-CAS improves the function of HepG2 cells. This system could therefore be applied to drug metabolism assay in the development of new drugs.

Preparation and biological evaluation of hydroxyapatite-coated nickel-free high-nitrogen stainless steel

Abstract Calcium phosphate was formed on nickel-free high-nitrogen stainless steel (HNS) by chemical solution deposition. The calcium phosphate deposition was enhanced by glutamic acid covalently immobilized on the surface of HNS with trisuccinimidyl citrate as a linker. X-ray diffraction patterns and Fourier transform infrared spectra showed that the material deposited on glutamic acid-immobilized HNS within 24 h was low-crystallinity calcium-deficient carbonate-containing hydroxyapatite (HAp). The biological activity of the resulting HAp-coated HNS was investigated by using a human osteoblast-like MG-63 cell culture. The HAp-coated HNS stimulated the alkaline-phosphate activity of the MG-63 culture after 7 days. Therefore, HAp-coated HNS is suitable for orthopedic devices and soft tissue adhesion materials.

Induction of hepatocyte functional protein expression by submicron/nano-patterning substrates to mimic in vivo structures

To investigate the influence of bio-inspired metallic superficial topography on the cellular behaviour of a hepatocyte cell line, TiO2 nanopatterns with diversified shapes and heterotropic lateral dimensions were fabricated using electron beam lithography and atomic layer deposition. The dimensional uniformity and shape diversity of the nanopatterns were confirmed using scanning electron microscopy and atomic force microscopy. These topographical nanocues provide good tools for controlling and regulating multiple hepatocellular functions. The expressions of functional proteins such as albumin, transferrin and cytochrome P450 were tested as functional markers. In addition, the change in cellular orientation, cell alignment and native extracellular matrix (ECM) assembly induced by these well-defined nanotopographies were observed. Twelve hours after cell seeding, TiO2 nanogratings with a lateral dimension of 240 nm showed a higher degree of functional protein expression compared to other nanotopographical substrates and a flat surface. These findings suggest that the TiO2 surface resembles a hierarchically-extended collagen nanofibrillar surface and could be recognized by hepatocytes, allowing the proper cytoskeletal orientation and cellular integrity. This TiO2 nanopattern with a specific shape and dimension (240 nm) might therefore emulate ECM biophysical cues, and the intrinsic topography of TiO2 surfaces might evoke enhanced cellular responses. These unique surfaces could be further exploited for tissue engineering and bioreactor technology.

Silver nanoparticles reduce the apoptosis induced by tumor necrosis factor-α

ABSTRACT Silver nanoparticles (AgNPs) are widely known to have anti-inflammatory properties, but the exact mechanism underlying this anti-inflammatory effect is not clearly understood. Tumor necrosis factor-α (TNFα) is a major pro-inflammatory cytokine that is expressed in the early stage of cell inflammation and induces apoptosis by several known pathways. Our study aimed to investigate the effect of AgNPs on the response of lung epithelial cells to TNFα and the molecular mechanism of this response. Lung epithelial cell line NCI-H292 cells were exposed to AgNPs (5 µg/mL) and/or TNFα (20 ng/mL) for 24 h, then cellular uptake was analyzed using flow cytometry. Our results showed that AgNPs were taken up by cells in a dose-dependent manner and that the cellular uptake ratio of AgNPs was significantly increased in the presence of TNFα. Apoptosis assays indicated that exposure to AgNPs significantly decreased the apoptotic effect of TNFα. Confocal microscopy was used to localize the tumor necrosis factor receptor 1 (TNFR1) and revealed that TNFR1 localized on the surface of cells exposed to TNFα. In contrast, TNFR1 localized inside cells exposed to both AgNPs and TNFα, with very few receptors scattered on the cell membrane. The results indicated that AgNPs reduced the cell surface TNFR1 expression level. The results suggested that the reduction of surface TNFR1 reduced cellular response to TNFα, resulting in an anti-apoptotic effect. GRAPHICAL ABSTRACT

PEGylated TiO2 nanoparticles mediated inhibition of cell migration via integrin beta 1

Abstract Nanoparticles (NPs) elicit various physiological responses in cellular environment, and the effect of NPs on cell migration is of high interest. In this work, the effects of NPs on cell migration and their possible mechanisms were studied. Here, we showed that after exposure to pegylated titanium dioxide nanoparticles (TiO2-PEG NPs, where PEG stands for the polyethylene glycol), NCI-H292 cells exhibited slower migration than control cells. Furthermore, larger NPs inhibited cell migration much stronger than smaller NPs. Following NP exposure, the cells showed decreased expression of integrin beta 1 and phosphorylated focal adhesion kinase (pFAK), and disrupted F-actin structures. We demonstrated that a possible mechanism involved NP-mediated promotion of the lysosomal degradation of integrin beta 1, thus leading to reduced expression of pFAK and cytoskeletal disruption and inhibited cell migration. Therefore, our results showed that inhibition of NCI-H292 cell migration by NPs is mediated through integrin beta 1, which provides useful information for the application of NPs in cancer therapy and related fields.