Novel Silver Complexes Based on Phosphanes and Ester Derivatives of Bis(pyrazol-1-yl)acetate Ligands Targeting TrxR: New Promising Chemotherapeutic Tools Relevant to SCLC Management

Abstract

Bis(pyrazol-1-yl)acetic acid (HC(pz)₂COOH) and bis(3,5-dimethyl-pyrazol-1-yl)acetic acid (HC(pz^Me2)₂COOH) were converted into the methyl ester derivatives 1 (L^OMe) and 2 (L^2OMe), respectively, and were used for the preparation of silver(I) complexes 3-5. The Ag(I) complexes were prepared by the reaction of AgNO₃ and 1,3,5-triaza-7-phosphaadamantane (PTA) or triphenylphosphine (PPh₃) with L^OMe and L^2OMe in methanol solution. All Ag(I) complexes showed a significant in vitro antitumor activity, proving to be more effective than the reference drug cisplatin in the in-house human cancer cell line panel containing examples of different solid tumors. Compounds were particularly effective against the highly aggressive and intrinsically resistant human small-cell lung carcinoma (SCLC) cells, either in 2D and 3D cancer cell models. Mechanistic studies revealed their ability to accumulate into cancer cells and to selectively target Thioredoxin (TrxR), thus leading to redox homeostasis unbalance and ultimately inducing cancer cell death through apoptosis.

Keywords: TrxR; anticancer activity; bis(pyrazolyl)acetate ligands; oxidative stress; silver.

Scheme 1

Chemical structures of complexes 3–5.

Figure 1

Cellular uptake and stability studies. (A) Intracellular accumulation of silver(I) complexes 3–5. U1285 cells were incubated with 2 μM of silver(I) complexes for 24 h, and cellular silver content was detected by GF-AAS analysis. Error bars indicate the standard deviation. ** p < 0.01 compared with control. (B) Stability studies. All complexes were dissolved at 50 μM in 0.5% DMSO/RPMI. UV–visible spectra were recorded at t = 0 min, t = 24 h, t = 48 h and t = 72 h.

Figure 2

TrxR inhibition. (A) TrxR1 activity was assayed by measuring NADPH-dependent reduction of DTNB at 412 nm as described in the Experimental Section. (a) 50% inhibition concentration (IC₅₀), Error bars indicate S.D. (B) U1285 cells were incubated for 24 h with the tested compounds (2 µM). Subsequently, cells were washed twice with PBS and lysed. TrxR activity was tested by measuring NADPH-dependent reduction of DTNB at 412 nm. Error bars indicate SD. * p < 0.05; ** p < 0.01.

Figure 3

Effects on sulfhydryl content and ROS production. (A) Sulfhydryl content in U1285-treated cancer cells incubated for 24 h with tested compounds. The sulfhydryl group amount was determined by the DTNB assay. Error bars indicate S.D. * p < 0.05, ** p < 0.01. (B) Effect of silver(I) compounds on hydrogen peroxide formation in U1285 cells. Cells were pre-incubated in PBS/10 mM glucose medium for 20 min at 37 °C in the presence of 10 μM CM–DCFDA and then treated with increasing concentrations of the tested compounds.

Figure 4

Antimitochondrial effect. (A) U1285 cells were treated for 24 h with increasing concentrations of the tested complexes or CCCP (3 μM). The percentage of cells with hypopolarized mitochondrial membrane potential was determined by Mito-ID^® Membrane Potential Kit. ** p ≤ 0.01. Error bars indicate S.D. (B) TEM analysis. Transmission electron micrographs of untreated U1285 cells or 24 h treated cells with IC₅₀ concentrations of complex 3 or complex 4.

Figure 5

Hoechst staining (magnification, ×20) of U1285 cells incubated for 48 h with IC₅₀ doses of 3–5.