Effects of internalized gold nanoparticles with respect to cytotoxicity and invasion activity in lung cancer cells

Abstract

The effect of gold nanoparticles on lung cancer cells is not yet clear. In this study, we investigated the cytotoxicity and cell invasion activity of lung cancer cells after treatment with gold nanoparticles and showed that small gold nanoparticles can be endocytosed by lung cancer cells and that they facilitate cell invasion. The growth of A549 cells was inhibited after treatment with 5-nm gold nanoparticles, but cell invasion increased. Endocytosed gold nanoparticles (size, 10 nm) notably promoted the invasion activity of 95D cells. All these effects of gold nanoparticles were not seen after treatment with larger particles (20 and 40 nm). The enhanced invasion activity may be associated with the increased expression of matrix metalloproteinase 9 and intercellular adhesion molecule-1. In this study, we obtained evidence for the effect of gold nanoparticles on lung cancer cell invasion activity in vitro. Moreover, matrix metalloproteinase 9 and intercellular adhesion molecule-1, key modulators of cell invasion, were found to be regulated by gold nanoparticles. These data also demonstrate that the responses of the A549 and 95D cells to gold nanoparticles have a remarkable relationship with their unique size-dependent physiochemical properties. Therefore, this study provides a new perspective for cell biology research in nanomedicine.

Figure 1. Characterization of the differently sized Au-NPs.

Transmission electron microscopy (TEM) images of Au-NPs with diameters of (A) 5 nm, (B) 10 nm, (C) 20 nm, or (D) 40 nm. The insets show high-resolution images. Scale bars, 20 nm and 100 nm (as marked). E: UV-vis spectra of the Au-NPs with 5-nm, 10-nm, 20-nm, and 40-nm diameters.

Figure 2. Transmission electron microscopy (TEM) images of Au-NPs trapped inside the cells.

TEM images at 200-nm magnification showing the internalization of 25 µg/mL Au-NPs with various sizes (5 nm, 10 nm, 20 nm, and 40 nm) into (A) A549 and (B) 95D cells after 48 h of treatment.

Figure 3. Comparison of the cytotoxicity of the A549 and 95D cell lines exposed to the Au-NPs.

The cell lines A549 (A) and 95D (B) in the logarithmic growth phase were exposed to Au-NPs for 24 h, 48 h, and 72 h. Cell viability was calculated as the percentage of the viable cells compared to the untreated controls. Each result represents the mean viability ± standard deviation (SD) of three independent experiments. Apoptosis (C–D) and cell cycle (E–H) analyses of A549 and 95D cells treated with differently sized Au-NPs. Error bars indicate SD values from four independent experiments. *P<0.05, **P<0.01, ***P<0.001.

Figure 4. Optical images and fluorescence measurements of cell invasion on Au-NP treatment.

The images represent A549 cells (A) and 95D cells (B) that have crossed the pores of the Matrigel invasion chamber and corresponding fluorescence intensity results. *P<0.05. Error bars indicate the SD values from three independent experiments.

Figure 5. Fold changes in mRNA expression tested by qRT-PCR in A549 and 95D cells treated with differently sized nanoparticles.

Results of qRT-PCR for (A–B) A549 and (C–D) 95D cells after 48-h incubation with 5-nm, 10-nm, 20-nm, or 40-nm Au-NPs. *P<0.05, ***P<0.001 vs. control. Error bars indicate the SD values from three independent experiments.

Figure 6. Expression levels of MMP9 in A549 and 95D cells after Au-NP treatment for 48 h.

Quantification of the expression of MMP9 in the cell lysate (A–B) and the supernatant (C–D) of A549 and 95D cells, respectively, was conducted by using the Luminex technology. *P<0.05. Error bars indicate the SD values from three independent experiments.

Figure 7. Western blot analysis of the expression of MMP9 and ICAM-1 in A549 and 95D cells.

Representative results showing the effect of Au-NPs treatment on the expression of MMP9 and ICAM-1 in A549 (A) and 95D (B) cells. Relative band density of MMP9 and ICAM-1 to the mean value of the control group (C–H). Values are expressed as relative intensity normalized to GAPDH intensity and are given as mean ± SD from three independent experiments. *P<0.05, **P<0.01, ***P<0.001 vs. control.