Enhanced Cytotoxic Effect of Doxorubicin Conjugated to Glutathione-Stabilized Gold Nanoparticles in Canine Osteosarcoma-In Vitro Studies

Abstract

Osteosarcoma (OSA) is the most common malignant bone neoplasia in humans and dogs. In dogs, treatment consists of surgery in combination with chemotherapy (mostly carboplatin and/or doxorubicin (Dox)). Chemotherapy is often rendered ineffective by multidrug resistance. Previous studies have revealed that Dox conjugated with 4 nm glutathione-stabilized gold nanoparticles (Au-GSH-Dox) enhanced the anti-tumor activity and cytotoxicity of Dox in Dox-resistant feline fibrosarcoma cell lines exhibiting high P-glycoprotein (P-gp) activity. The present study investigated the influence of Au-GSH-Dox on the canine OSA cell line D17 and its relationship with P-gp activity. A human Dox-sensitive OSA cell line, U2OS, served as the negative control. Au-GSH-Dox, compared to free Dox, presented a greater cytotoxic effect on D17 (IC₅₀ values for Au-GSH-Dox and Dox were 7.9 μg/mL and 15.2 μg/mL, respectively) but not on the U2OS cell line. All concentrations of Au-GSH (ranging from 10 to 1000 μg/mL) were non-toxic in both cell lines. Inhibition of the D17 cell line with 100 μM verapamil resulted in an increase in free Dox but not in intracellular Au-GSH-Dox. The results indicate that Au-GSH-Dox may act as an effective drug in canine OSA by bypassing P-gp.

Keywords: P-glycoprotein; dogs; doxorubicin; flow cytometry; gold; humans; nanoparticles; osteosarcoma.

Figure 1

Correlation between the logarithm of Dox (red line) and Au-GSH-Dox (black line) doses and cell viability (measured by MTT assay) for D17 (A) and U2OS (B) cell lines.

Figure 2

Statistical analyses of the percentage of cells in apoptosis and necrosis measured with annexin-V and PI assay on D17 (A) and U2OS (B) cell lines treated with Au-GSH-Dox and Dox in an IC₅₀ Au-GSH-Dox dose measured with the MTT assay. *** p ≤ 0.001 was interpreted as highly significant.

Figure 3

Scatter diagram of D17 (A) and U2OS (B) cells treated with tested substances in annexin-V and PI assay, measured by flow cytometry (treated with Au-GSH-Dox and Dox in an IC₅₀ Au-GSH-Dox dose measured by MTT assay).

Figure 4

Effect of the tested substances (Au-GSH-Dox and Dox) on the mortality of the tested cell lines: D17 (A) and U2OS (B). ** p ≤ 0.01 and *** p ≤ 0.001 were interpreted as highly significant.

Figure 5

Phase-contrast microscopy images of D17 and U2OS OSA cells treated with 5 μg/mL of Dox (A,E), 5 μg/mL of Au-GSH-Dox (B,F), 100 μg/mL of Au-GSH (as the Dox/Au-GSH concentration ratio in Au-GSH-Dox was 1:20) (C,G), and untreated (negative control) (D,H). 4× magnification, scale bar 200 μm.

Figure 6

Fluorescence of rhodamine 123 with or without verapamil in treated D17 (A) and U2OS (B) cell lines.

Figure 7

Fluorescence of D17(A) and U2OS (B) after treatment with Au-GSH-Dox or Dox alone with or without verapamil, analyzed by flow cytometry.