Continuous Long-Term Exposure to Low Concentrations of MWCNTs Induces an Epithelial-Mesenchymal Transition in BEAS-2B Cells

Abstract

In the field of nanotechnology, the use of multi-walled carbon nanotubes (MWCNTs) is growing. Pulmonary exposure during their production, use, and handling is raising concerns about their potential adverse health effects. The purpose of this study is to assess how the physical characteristics of MWCNTs, such as diameter and/or length, can play a role in cellular toxicity. Our experimental design is based on the treatment of human bronchial epithelial cells (BEAS-2B) for six weeks with low concentrations (0.125-1 µg/cm²) of MWCNTs having opposite characteristics: NM-403 and Mitsui-7. Following treatment with both MWCNTs, we observed an increase in mitotic abnormalities and micronucleus-positive cells. The cytotoxic effect was delayed in cells treated with NM-403 compared to Mitsui-7. After 4-6 weeks of treatment, a clear cellular morphological change from epithelial to fibroblast-like phenotype was noted, together with a change in the cell population composition. BEAS-2B cells underwent a conversion from the epithelial to mesenchymal state as we observed a decrease in the epithelial marker E-cadherin and an increased expression of mesenchymal markers N-cadherin, Vimentin, and Fibronectin. After four weeks of recovery, we showed that the induced epithelial-mesenchymal transition is reversible, and that the degree of reversibility depends on the MWCNT.

Keywords: BEAS-2B cells; MWCNTs; epithelial-mesenchymal transition; nanotoxicology.

Figure 1

Scheme of the treatment schedule for BEAS-2B cells. (A) The cells were seeded at 3500 cells/cm² on day 0. Cells were treated with 0.125, 0.25 and 0.5 μg/cm² of Mitsui-7 or 0.25, 0.5 and 1 μg/cm² of NM-403 twice a week for 6 weeks followed by 4 weeks without treatment. (B) Micronucleus assay, abnormal mitoses, cell proliferation and cytotoxicity were evaluated the first four weeks (W1 to W4). Cell morphology was observed weekly from the 1st week of treatment to the 4th week of the recovery period. Analysis of EMT markers using RT-qPCR and Flow Cytometry was conducted after 4 to 6 weeks of treatment (W4 to W6) and after the 4th week of the recovery period (RW4). EMT: epithelial-mesenchymal transition, RW: recovery week, W: week.

Figure 2

Antiproliferative and cytotoxic effect of MWCNTs. BEAS-2B cells were treated with Mitsui-7 (upper) or NM-403 (lower) at the indicated concentrations for 4 weeks. (A) Proliferation was assessed using a NucLight probe, which labeled total cells. (B) Cytotoxicity assay was assessed using Cytotox and NucLight probes and the ratio of dead cells/total cells was determined. Data were normalized to the first point of acquisition after addition of the dyes (4 h). The graphs are representative of three independent experiments.

Figure 3

Abnormal mitoses induced by treatment with Mitsui-7 and NM-403. (A) The pictures represent a normal mitosis and abnormal mitoses with a monopolar or multipolar spindle. (B) The cells were treated with Mitsui-7 (upper panel) or NM-403 (lower panel) at the indicated concentrations for 4 weeks. The cells were immunolabeled with an anti-α-Tubulin antibody and the nuclei were stained with DAPI. Mitotic spindles were analysed within 200 dividing cells per sample. The histograms indicate the percentage of abnormal mitoses: dashed bars for multipolar and solid bars for monopolar mitoses. A treatment with vanadium pentoxide (V₂O₅, 1.25 µg/cm²) for 24 h was used as a positive control. The histograms represent the mean ± standard error of the mean (SEM) of three independent experiments. *** p < 0.001 significantly different from the control.

Figure 4

Treatment with Mitsui-7 and NM-403 increased the occurrence of micronuclei. The cells were treated with (A) Mitsui-7 (0.125, 0.25 and 0.5 μg/cm²) or (B) NM-403 (0.25, 0.5 and 1 μg/cm²) for 4 weeks. The cells were immunolabeled with an anti-α-Tubulin antibody and the nuclei were stained with DAPI and 2000 cells were analysed in each sample. The fluorescent microscopy images represent a micronucleus following DAPI staining (white arrow). The histograms indicate the percentage of micronucleus-positive cells. A treatment with vanadium oxide (V₂O₅, 1.25 µg/cm²) for 24 h was used as a positive control. The histograms represent the mean ± standard error of the mean (SEM) of three independent experiments. *** p < 0.001 significantly different from the control.

Figure 5

Morphological changes of BEAS-2B cells induced by Mitsui-7 and NM-403. The cells were treated with Mitsui-7 (0.125, 0.25 and 0.5 μg/cm²) or NM-403 (0.25, 0.5 and 1 μg/cm²) for 6 weeks. Cell morphology was observed weekly with an optical microscope at the 10× objective. Representative pictures of morphological changes are presented from normal to needle-shaped cells (upper panel). The heatmap represents the morphological changes, light blue for cells with a normal shape to dark blue for cells with a needle shape (lower panel). The results are representative of at least three independent experiments. W: weeks.

Figure 6

Mitsui-7 and NM-403 induced the selection of a cell population. The cells were treated with (A) Mitsui-7 (0.125, 0.25 and 0.5 μg/cm²) or (B) NM-403 (0.25, 0.5 and 1 μg/cm²) and their size and granulometry were analysed using flow cytometry. Based on the SSC:FSC profiles, two cell populations were determined: those of a small size and low granulometry (small) and those of a large size and high granulometry (large). The SSC:FSC profiles from W1, W4, W5 and W6 are represented for Mitsui-7 0.25 µg/cm² and NM-403 0.25 µg/cm². The histograms represent the mean ± standard error of the mean (SEM) of the quantification of the two cell populations for three independent experiments. ** p < 0.01, *** p < 0.001 significantly different from the control.

Figure 7

MWCNTs-induced EMT marker expression. RNAs from total cells treated with the indicated concentration of Mitsui-7 (left) or NM-403 (right) were extracted and gene expression was analysed using RT-qPCR. The epithelial marker E-cadherin (cdh1) and mesenchymal markers N-cadherin (cdh2), Vimentin (vim) and Fibronectin (fn1) were evaluated after 4, 5 and 6 weeks of treatment. The histograms represent the mean ± standard error of the mean (SEM) for three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 significantly different from the control.

Figure 8

MWCNTs reduced E-cadherin in BEAS-2B cells. (A) The cells were treated with vehicle, 0.25 µg/cm² of Mitsui-7 or 0.5 µg/cm² of NM-403 for 6 weeks. The cells were immunolabeled with an anti-E-cadherin antibody and the nuclei were stained with DAPI. (B) Cells treated with the indicated concentrations of Mitsui-7 or NM-403 for 4 to 6 weeks were labeled with the anti-E-cadherin antibody and analysed using flow cytometry. The extracellular marker E-cadherin was quantified in the population of small cells and the bar graph represents the ratio of mean fluorescence intensity/total number of cells reported in the vehicle. The histograms represent the mean ± standard error of the mean (SEM) for three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 significantly different from the control.

Figure 9

The MWCNTs-induced EMT is reversible. After the 6 weeks of treatment with Mitsui-7 (0.125, 0.25 and 0.5 μg/cm²) or NM-403 (0.25, 0.5 and 1 μg/cm²), the cells were cultured without treatment for 4 weeks (RW1-RW4). (A) The heatmap represents the morphological changes observed in cells during the 4 weeks of the recovery period from needle shape (dark blue) to normal shape (light blue). (B) The two cellular populations (small and large cells) were analysed using flow cytometry and determined based on SSC:FSC profiles during the RW4. The extracellular marker E-cadherin was quantified in the population of small cells using flow cytometry and the bar graph represents the ratio of mean fluorescence intensity/total number of cells reported to the vehicle. The histograms represent the mean ± standard error of the mean (SEM) for three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 significantly different from the control.