Elsevier

Toxicology Letters

Volume 155, Issue 1, 15 January 2005, Pages 73-85
Toxicology Letters

Effect of single wall carbon nanotubes on human HEK293 cells

https://doi.org/10.1016/j.toxlet.2004.08.015 Get rights and content

Abstract

The influence of single-walled carbon nanotubes (SWCNTs) on human HEK293 cells is investigated with the aim of exploring SWCNTs biocompatibility. Results showed that SWCNTs can inhibit HEK293 cell proliferation, decrease cell adhesive ability in a dose- and time-dependent manner. HEK293 cells exhibit active responses to SWCNTs such as secretion of some 20–30 kd proteins to wrap SWCNTs, aggregation of cells attached by SWCNTs and formation of nodular structures. Cell cycle analysis showed that 25 μg/ml SWCNTs in medium induced G1 arrest and cell apoptosis in HEK293 cells. Biochip analysis showed that SWCNTs can induce up-regulation expression of cell cycle-associated genes such as p16, bax, p57, hrk, cdc42 and cdc37, down-regulation expression of cell cycle genes such as cdk2, cdk4, cdk6 and cyclin D3, and down-regulation expression of signal transduction-associated genes such as mad2, jak1, ttk, pcdha9 and erk. Western blot analysis showed that SWCNTs can induce down-regulation expression of adhesion-associated proteins such as laminin, fibronectin, cadherin, FAK and collagen IV. These results suggest that down-regulation of G1-assoicated cdks and cyclins and upregulation of apoptosis-associated genes may contribute to SWCNTs induced G1 phase arrest and cell apoptosis. In conclusion, SWCNTs can inhibit HEK293 cells growth by inducing cell apoptosis and decreasing cellular adhesion ability.

Introduction

Carbon nanotubes, as a class of stiff, stable and hollow nanomaterials with many unique properties such as mechanical, physical and chemical properties, have been being explored application in biomedical engineering and medical chemistry (Baughman et al., 2002, Bianco and Prato, 2003, Pantarotto et al., 2004). For example, carbon nanotubes have been used as AFM tip to obtain atomic-resolution imaging of biological molecules such as DNA and proteins (Nagao et al., 2000, Hafner et al., 2001). Our research also show that carbon nanotubes can be filled with DNA or peptide molecules and have highly potential in gene or peptide storage and delivery system in molecular therapy of diseases (Gao et al., 2003, Cui and Gao, 2003, Cui et al., 2004a). Carbon nanotubes can be used to fabricate nanomotors, which likely enter inside cells to treat diseases. So far, it has becoming a focus to investigate the influence of carbon nanotubes and associated nanomaterials or nanodevices on human cells and environment. Carbon nanotubes can be functionalized to achieve improved properties and functions such as biocompatibility and biomolecular recognition capabilities (Shim et al., 2002, Bahr and Tour, 2002). The potential with which carbon nanotubes can be applied in biomedical engineering and medicinal chemistry is highly dependent upon their biocompatibility. Carbon nanotubes exhibit cytotoxicity to human keratinocyte cells (Robert, 2003, Shvedova et al., 2003), can inhibit the growth of embryonic rat-brain neuron cells (Mattson et al., 2001) and induce the formation of mouse-lung granulomas (Chan et al., 2003, Warheit et al., 2004, Maynard et al., 2004, Lam et al., 2004). However, so far few reports are closely associated with mechanism of effect of carbon nanotubes on human normal or tumor or embryonic tissue cells. Therefore, investigating effect of carbon nanotubes on human cells and their interaction mechanism is of very importance.

Here, we selected human HEK293 cells as research target, investigated the interaction between SWCNTs and HEK293 cells by morphological observation, Western blot, flow cytometry, immunofluorescent analysis and biochip analysis. Result shows that SWCNTs can inhibit the proliferation of human HEK293 cells, induce cell apoptosis, decrease cell adhesive ability, and cause the cells to secrete some 20–30 kd proteins, which wrap SWCNTs into nodular structures and isolate SWCNTs-attached cells from the main cell populations. A model of signal transduction and cellular pathways of SWCNTs-cell interaction was proposed. The main purpose of current study was to explore the effect of SWCNTs on human HEK293 cells and potential biochemical mechanism, laying foundation for further exploring carbon nanotubes application on molecular therapy of diseases in near future.

Section snippets

Single-walled carbon nanotubes and antibodies

Single-walled carbon nanotubes (SWCNTs) were purchased from Carbon Nanotechnologies, Inc. (CAS no. 7782-42-5). Monoclonal anti-human fibronectin antibody (product no. F7387), anti-focal adhesion kinase (pp125FAK) antibody (product no. F2918), anti-pan cadherin antibody (product no. C3678), monoclonal anti-collagen type IV clone COL-94 (product no. 1926), monoclonal anti-laminin clone lam-89 (product no. L8271), anti-cyclin D3 antibody (product no. C7214), monoclonal anti-β-actin clone AC-15

Effect of SWCNTs on the viability and proliferation of HEK293 cells

Since cell viability is positively correlated with the degree of MTT reduction, the cell viability of SWCNTs-treated HEK293 cells were evaluated by using MTT reduction assays. As indicated in Fig. 1A, treatment of HEK293 cells with various concentrations (0.78125 μg/ml, 1.5625 μg/ml, 3.125 μg/ml, 6.25 μg/ml, 12.5 μg/ml, 25 μg/ml, 50 μg/ml, 100 μg/ml, 150 μg/ml and 200 μg/ml) of SWCNTs caused a time- and dose-dependent decrease in cell viability relative to the control culture.

As indicated in Fig. 1B,

Discussion

Carbon nanotubes, because of their unique properties, own great potential applications on biomedical engineering and medical chemistry. For example, carbon nanotubes own catalytic function, which possibly affect cellular metabolism (Cui et al., 2004b). Carbon nanotubes can be filled with target DNA molecules or peptides, which has high potential in delivering target DNA molecules or peptides into special tissue region to treat the diseases (Guo et al., 1998, Gao et al., 2003, Cui et al., 2004a,

Acknowledgment

We thank Dr. Heinz Schwarz of Max Planck Institute for Development Research for help on immunostaining experiments.

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