Cellular Metabolic Responses to Copper Nanoparticles: Comparison Between Normal and Breast Cancer Cells

Abstract

The use of copper nanoparticles (CuNPs) seems to be an alternative therapeutic strategy for cancer therapy due to low-cost synthesis and anticancer activity. In this work, CuNPs' effects were tested in various concentrations on two types of cells: mesenchymal stem cells (MSCs) and a breast cancer cell line, SKBR3. The concentrations (0.25 mM, 0.5 mM, 1 mM and 2 mM) were first tested on an impedance-based cytotoxicity assay and then used in further cellular metabolic assays. Next, several techniques were applied to test the chosen concentrations: assessment of apoptosis, intracellular reactive oxygen species (ROS) levels, oxidative stress-related gene expression, assessment of mitochondrial respiration and fatty acid methyl ester (FAME) profile evaluation. The higher CuNP concentrations tested on the SKBR3 cell line showed a dose-dependent decrease in the cell index. SKBR3 cells displayed increased CAT and SOD expression, revealed by strong dose-dependent fluorescence. Annexin/PI staining confirmed increased SKBR3 cell death induced by the higher doses of CuNPs. SKBR3 revealed higher baseline respiratory capacity compared to MSCs. Fatty acid methyl esters (FAMEs) are in higher abundance in MSCs compared to the SKBR3 cell line. The different metabolic response in the tested cells to the CuNPs' presence could help establish a future personalized treatment for breast cancer patients.

Keywords: in vitro toxicity; metabolomics; nanomaterials; oxidative stress.

Figure 1

(A) SEM picture of CuNPs; (B) energy-dispersive X-ray spectrum (EDX) of the nanomaterial; (C) experimental determination of the mean diameter for CuNPs.

Figure 2

Theoretical evaluation of the CuNPs’ diameter.

Figure 3

Real-time monitoring of cell index in MSCs (A) and SKBR3 cells (B) using the xCELLigence system. Cells were treated with CuNPs at different concentrations: 0.25 mM (light blue), 0.5 mM (magenta), 1 mM (dark blue) and 2 mM (green) for 7 days. Control is plotted in red.

Figure 4

Annexin V and PI staining in MSCs treated with different concentrations of CuNPs. The lower left quadrant represents live cells (Annexin V, PI negative), the lower right quadrant represents early apoptotic cells (Annexin V positive, PI negative), the upper right quadrant represents late apoptotic cells (Annexin V and PI positive), and the necrotic or dead cells (Annexin V negative and PI positive) are presented in the upper left quadrants. The bar graph (mean ± SD from n = 3 independent experiments) summarizes the quantitative distribution of viable, apoptotic and necrotic cells across treatments, indicating a dose-dependent cytotoxic effect at higher CuNP concentrations. Statistical significance is denoted as follows: *** p < 0.001.

Figure 5

Annexin V and PI staining in SKBR3 cells treated with different concentrations of CuNPs. Representative dot plots illustrate the distribution of the viable, early apoptotic, late apoptotic and necrotic populations under each treatment conditions. The bar graph (mean ± SD from n = 3 independent experiments) summarizes the quantitative distribution of viable, apoptotic and necrotic SKBR3 cells following CuNP exposure, demonstrating a clear dose-dependent cytotoxic effect and increased sensitivity to higher CuNP concentrations compared to MSCs. Statistical significance is denoted as follows: * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 6

Intracellular ROS levels in MSCs and SKBR3 cells following CuNP treatments. Representative bright-field images for MSCs (A) and SKBR3 cells (C) and fluorescence images for MSCs and SKBR3 cells using the DCFH-DA fluorescence probe (B,D). Cells were exposed to increasing concentrations of CuNPs. Scale bar: 50 µm.

Figure 7

Antioxidant gene expression and cellular response to CuNPs. The expression of oxidative stress-related genes (PPARγ, SOD and CAT) was assessed by quantitative PCR, with GAPDH as the internal reference gene for normalization. Data are presented as the mean ± SEM from three independent experiments. Statistical significance is denoted as follows: *** p < 0.001. The oxidative stress-related genes (PPARγ, SOD and CAT) were represented via agarose gel electrophoresis (Figure S1).

Figure 8

Oxygen consumption rates in MSCs and SKBR3 cells following CuNP treatments, assessed using the Oroboros 2k high-resolution spirometry system. The figure illustrates the effects of CuNPs on routine respiration (R), leak respiration (L), maximal respiratory capacity (M) and oxidative phosphorylation capacity (OXPHOS). Representative oxygraph depicting oxygen consumption dynamics and basal respiration are shown in the Supplementary Files (Figures S2 and S3). Statistical significance is denoted as follows: ** p < 0.01 and *** p < 0.001.

Figure 9

The effect of CuNPs on the proportion of FAMEs in the MSCs.

Figure 10

The effect of CuNPs on the proportion of FAMEs in the SKBR3 cell line.

Figure 11

The effect of increasing CuNP concentrations on saturated (SFA), mono- (MUFA) and poly-unsaturated (PUFA) FAME levels in MSCs and SKBR3 cells.