Antitumor Activity of Metal-Chelating Compound Dp44mT Is Mediated by Formation of a Redox-Active Copper Complex That Accumulates in Lysosomes

Abstract

The metal-chelating compound Dp44mT is a di-2-pyridylketone thiosemicarbazone (DpT) which displays potent and selective antitumor activity. This compound is receiving translational attention, but its mechanism is poorly understood. Here, we report that Dp44mT targets lysosome integrity through copper binding. Studies using the lysosomotropic fluorochrome acridine orange established that the copper-Dp44mT complex (Cu[Dp44mT]) disrupted lysosomes. This targeting was confirmed with pepstatin A-BODIPY FL, which showed redistribution of cathepsin D to the cytosol with ensuing cleavage of the proapoptotic BH3 protein Bid. Redox activity of Cu[Dp44mT] caused cellular depletion of glutathione, and lysosomal damage was prevented by cotreatment with the glutathione precursor N-acetylcysteine. Copper binding was essential for the potent antitumor activity of Dp44mT, as coincubation with nontoxic copper chelators markedly attenuated its cytotoxicity. Taken together, our studies show how the lysosomal apoptotic pathway can be selectively activated in cancer cells by sequestration of redox-active copper. Our findings define a novel generalized strategy to selectively target lysosome function for chemotherapeutic intervention against cancer. Cancer Res; 71(17);

The Metastasis Suppressor, N-MYC Downstream-regulated Gene-1 (NDRG1), Down-regulates the ErbB Family of Receptors to Inhibit Downstream Oncogenic Signaling Pathways

“…In fact, although EGFR inhibitors have shown promise for the treatment of some tumors, their effects are inconsistent and largely affected by the many different mutations and alterations of EGFR encountered in cancer patients (46). In contrast, the thiosemicarbazone, Dp44mT, was found to overcome resistance to many antitumor agents (23,69). Moreover, in this study, we demonstrate that the pancreatic PANC-1 and MIAPaCa-2 as well as the colon HT-29 and HCT-116 cells were far more sensitive to Dp44mT and DpC when compared with the clinically used EGFR inhibitor, erlotinib (28,70).…”

“…The stoichiometries of ligand to metal used represent the Fe(III) and Cu(II) complexes observed with thiosemicarbazones of this class (21,38). It is notable that a DpCcopper complex concentration of 1 M was used, rather than the 10 M that was implemented for the DpC-iron complex, due to the high anti-proliferative activity of the copper complexes of this class of thiosemicarbazones (69). The metal ions, Fe(III) (as FeCl 3 ) and Cu(II) (as CuCl 2 ), were also implemented as relevant controls in these studies, as they were used to prepare the metal complexes.…”

The Anticancer Agent Di-2-pyridylketone 4,4-Dimethyl-3-thiosemicarbazone (Dp44mT) Overcomes Prosurvival Autophagy by Two Mechanisms

“…Assessment of Lysosomal Membrane Permeability-The distribution of acridine orange (Sigma-Aldrich) was assessed to determine the integrity of the lysosomal membrane as reported previously (27). After incubation with the examined agent, cells were incubated for 15 min at 37°C with acridine orange (20 nM) and washed three times with PBS.…”

“…Unlike DFO, Dp44mT forms redox-active iron or copper complexes that play key roles in its cytotoxic activity (20,(25)(26)(27)(28). In fact, Dp44mT was shown to be lysosomotropic and was retained within lysosomes due to their acidic pH and the ionization characteristics of Dp44mT (27). Within this organelle, Dp44mT forms cytotoxic iron or copper complexes that generate reactive oxygen species (ROS) that result in lysosomal membrane permeabilization and cell death (27).…”

“…Given that Dp44mT and DFO both localize to the lysosomal compartment and bind iron or copper (27,29), the effect of Dp44mT and DFO on autophagy is of interest. Because DFO is known to induce cell death (30) and Dp44mT is potently cytotoxic (20), it is of interest whether autophagy will play a prosurvival role (1,4).…”