Role of metalation in the topoisomerase IIα inhibition and antiproliferation activity of a series of α-heterocyclic-N4-substituted thiosemicarbazones and their Cu(II) complexes

Abstract

The topoisomerase-IIα inhibition and antiproliferative activity of α-heterocyclic thiosemicarbazones and their corresponding copper(II) complexes have been investigated. The Cu(II)(thiosemicarbazonato)Cl complexes were shown to catalytically inhibit topoisomerase-IIα at concentrations (0.3-7.2 μM) over an order of magnitude lower than their corresponding thiosemicarbazone ligands alone. The copper complexes were also shown to inhibit the proliferation of breast cancer cells expressing high levels of topoisomerase-IIα (SK-BR-3) at lower concentrations than cells expressing lower levels of the enzyme (MCF-7).

Figure 1

Agarose gel assay for Topo-IIα inhibition by Cu(TSC)Cl complexes and TSC ligands alone. In the Cu(TSC)Cl gel (A), 1 mM etoposide was employed as a positive control, and lanes 1–11 denote the reaction of Topo-IIα with supercoiled (sc) DNA in the presence of 10 μM Cu(Fp4mT)Cl, Cu(Fp4eT)Cl, Cu(Fp4ipT)Cl, Cu(Fp4alT)Cl, Cu(Ap4mT)Cl, Cu(Ap4eT)Cl, Cu(Fp4pyrrT)Cl, Cu(Fp4bzT)Cl, Cu- (FpT)Cl, Cu(Ap4alT)Cl, and Cu(Apz4mT)Cl. In the TSC gel (B), 100 μM etoposide was employed as a positive control, and lanes 1–11 denote the reaction of Topo-IIα with supercoiled DNA in the presence of 100 μM HFp4mT, HFp4eT, HFp4ipT, HFp4alT, HAp4mT, HAp4-eT, HFp4pyrrT, HFp4bzT, HFpT, HAp4alT, and HApz4mT.

Figure 2

Representative gel-based topoisomerase IIα inhibition assay illustrating the mechanism of Topo-IIα inhibition by Cu(Fp4alT)Cl. The top gel (A) was poststained with 5 μg/mL ethidium bromide in 1× TAE buffer, while the bottom gel (B) was electrophoresed with 5 μg/mL ethidium bromide within the gel. High concentrations of enzyme (4 units) and inhibitors (500 μM) were used to maximize the intensity of linear DNA bands (where present). Arrows mark representative linear DNA bands.

Figure 3

Western blot (left) of relative Topo-IIα expression in SK-BR-3 and MCF-7 cells. Quantiation (right) was normalized using actin loading control. Error bars are (1 standard deviation and are the result of six independent experiments.

Scheme 1

General Synthetic Route to the TSCs and Corresponding Cu^II(TSC)Cl Complexes

Chart 1

Structures of the Thiosemicarbazone Ligands and Their Cu^II(TSC)Cl Complexes^{a a}When the ligand is discussed alone in the text, an “H” is placed before the name (e.g., HFp4mT).

Chart 2

Topo-IIα Inhibition Activity (IC₅₀) and Antiproliferative Activity (GI₅₀) of the TSCs and Cu^II(TSC)Cl Complexes^{b a}The Topo-IIα inhibition IC₅₀ for etoposide, merbarone, and doxorubicin vary somewhat in the literature; in this chart, a range of concentrations and representative publications are presented. ^bTopo-IIα inhibition IC₅₀ values were determined via the described agarose gel supercoiled DNA relaxation assay employing a range of drug concentrations from 500 μM to 10 nM. Cell proliferation GI₅₀ values were determined via MTT assay with drug concentrations ranging from 500 μM to 10 nM. All experiments were performed in triplicate, and all values are shown with (1 SD.