Light Stability, Pro-Apoptotic and Genotoxic Properties of Silver (I) Complexes of Metronidazole and 4-Hydroxymethylpyridine against Pancreatic Cancer Cells In Vitro

Abstract

Antimicrobial properties of silver (I) ion and its complexes are well recognized. However, recent studies suggest that both silver (I) ion and its complexes possess anticancer activity associated with oxidative stress-induced apoptosis of various cancer cells. In this study, we aimed to investigate whether silver nitrate and its complexes with metronidazole and 4-hydroxymethylpyridine exert anticancer action against human pancreatic cancer cell lines (PANC-1 and 1.2B4). In the study, we compared decomposition speed for silver complexes under the influence of daylight and UV-A (ultraviolet-A) rays. We employed the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazonium bromide) assay to evaluate the cytotoxicity and the alkaline comet assay to determine genotoxicity of silver nitrate and its complexes. Flow cytometry and the Annexin V-FITC/PI apoptosis detection kit were used to detect the apoptosis of human pancreatic cancer cells. We found a dose dependent decrease of both pancreatic cancer cell line viability after exposure to silver nitrate and its complexes. The flow cytometry analysis confirmed that cell death occurred mainly via apoptosis. We also documented that the studied compounds induced DNA damage. Metronidazole and 4-hydroxymethylpyridine alone did not significantly affect viability and level of DNA damage of pancreatic cancer cell lines. Complex compounds showed better stability than AgNO₃, which decomposed slower than when exposed to light. UV-A significantly influences the speed of silver salt decomposition reaction. To conclude, obtained data demonstrated that silver nitrate and its complexes exerted anticancer action against human pancreatic cancer cells.

Keywords: 4-hydroxymethylpyridine; UV-A rays; apoptosis; complex; cytotoxicity; genotoxicity; light stability; medicinal chemistry; metronidazole; pancreatic cancer cells; silver (I).

Scheme 1

Synthesis of silver (I) complex of metronidazole.

Scheme 2

Synthesis of silver (I) complex of 4-hydroxymethylpyridine.

Figure 1

The cytotoxic activity of silver nitrate (AgNO₃), metronidazole, 4-hydroxymethylpyridine, ((MTZ)₂Ag)NO₃, and ((4-OHMePy)₂Ag)NO₃ against PANC-1 and 1.2B4 cells. The cells were treated with the tested compounds in the 0–300 µM concentration range, and cisplatin (0–100 µM) was employed as a positive control. After 72-h of incubation, cytotoxicity was determined using the MTT assay. Data are expressed as means ± SD from three to five independent experiments. * p < 0.05; ** p < 0.01, *** p < 0.001 in comparison to untreated cells (negative control).

Figure 2

(A) The level of DNA damage in PANC-1 and 1.2B4 cells exposed to silver nitrate (AgNO₃), metronidazole, 4-hydroxymethylpyridine, ((MTZ)₂Ag)NO₃, and ((4-OHMePy)₂Ag)NO₃ (0.0, 0.5, 0.75, 1.0, 5.0 µM) for 1 h at 37 °C. The DNA damage was determined by the alkaline comet assay. Treatment with 20 µM H₂O₂ for 10 min on ice served as a positive control. Data are expressed as mean ± SEM. * p < 0.05; ** p < 0.01, *** p < 0.001 in comparison to untreated cells (negative control). (B,C) The representative images of comets after exposure to the tested agents visualized by staining with 2 mg/mL 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) and observed under fluorescence microscope (Nikon, Tokyo, Japan) at 200× magnification.

Figure 2

(A) The level of DNA damage in PANC-1 and 1.2B4 cells exposed to silver nitrate (AgNO₃), metronidazole, 4-hydroxymethylpyridine, ((MTZ)₂Ag)NO₃, and ((4-OHMePy)₂Ag)NO₃ (0.0, 0.5, 0.75, 1.0, 5.0 µM) for 1 h at 37 °C. The DNA damage was determined by the alkaline comet assay. Treatment with 20 µM H₂O₂ for 10 min on ice served as a positive control. Data are expressed as mean ± SEM. * p < 0.05; ** p < 0.01, *** p < 0.001 in comparison to untreated cells (negative control). (B,C) The representative images of comets after exposure to the tested agents visualized by staining with 2 mg/mL 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) and observed under fluorescence microscope (Nikon, Tokyo, Japan) at 200× magnification.

Figure 2

(A) The level of DNA damage in PANC-1 and 1.2B4 cells exposed to silver nitrate (AgNO₃), metronidazole, 4-hydroxymethylpyridine, ((MTZ)₂Ag)NO₃, and ((4-OHMePy)₂Ag)NO₃ (0.0, 0.5, 0.75, 1.0, 5.0 µM) for 1 h at 37 °C. The DNA damage was determined by the alkaline comet assay. Treatment with 20 µM H₂O₂ for 10 min on ice served as a positive control. Data are expressed as mean ± SEM. * p < 0.05; ** p < 0.01, *** p < 0.001 in comparison to untreated cells (negative control). (B,C) The representative images of comets after exposure to the tested agents visualized by staining with 2 mg/mL 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) and observed under fluorescence microscope (Nikon, Tokyo, Japan) at 200× magnification.

Figure 3

The flow cytometric (Annexin V-FITC/PI) analysis of apoptosis of PANC-1 (A) and 1.2B4 (B) cells induced by the exposure to silver nitrate (AgNO₃), metronidazole, 4-hydroxymethylpyridine, ((MTZ)₂Ag)NO₃, and ((4-OHMePy)₂Ag)NO₃ (0.0, 5, 10, 25 µM) for 72 h. As a positive control 10 µM cisplatin was used. After treatment with the tested compounds, the cells were stained with Annexin V and propidium iodide (PI), left for 15 min in the dark and analyzed by flow cytometer. The percentages of cells in early (Annexin V+, PI−; lower right quadrant; F+– and late apoptotic-necrotic stages (Annexin V+, PI+; upper right quadrant; F++) are shown. The results are representative of three independent experiments.

Figure 3

The flow cytometric (Annexin V-FITC/PI) analysis of apoptosis of PANC-1 (A) and 1.2B4 (B) cells induced by the exposure to silver nitrate (AgNO₃), metronidazole, 4-hydroxymethylpyridine, ((MTZ)₂Ag)NO₃, and ((4-OHMePy)₂Ag)NO₃ (0.0, 5, 10, 25 µM) for 72 h. As a positive control 10 µM cisplatin was used. After treatment with the tested compounds, the cells were stained with Annexin V and propidium iodide (PI), left for 15 min in the dark and analyzed by flow cytometer. The percentages of cells in early (Annexin V+, PI−; lower right quadrant; F+– and late apoptotic-necrotic stages (Annexin V+, PI+; upper right quadrant; F++) are shown. The results are representative of three independent experiments.