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.
Highlights
Neoplastic cells have high requirements for iron (Fe) due to their generally higher rates of proliferation than normal cells (1)
We showed that apoptosis occurred via the mitochondrial pathway, where decreased Bcl[2] and increased Bax expression occurred along with holocytochrome c (h-cytc) release and caspase activation (6)
Our study suggests that these apoptotic events could be caused by Cu[Dp44mT]-induced redox stress that results in lysosomal membrane permeability (LMP), causing redistribution of lysosomal cathepsins to the cytosol (Fig. 4A) and concomitant cleavage of BH3-interacting domain death agonist (Bid) into its proapoptotic form (Fig. 4B)
Summary
Neoplastic cells have high requirements for iron (Fe) due to their generally higher rates of proliferation than normal cells (1). Neoplastic cells express enhanced transferrin receptor 1 (TfR1) levels relative to their normal counterparts (2) and take up Fe from transferrin (Tf) at a rapid rate (3), making them selectively sensitive to Fe chelation. Cancer cells take up more copper (Cu) than their normal counterparts, as this metal is essential for angiogenesis and metastasis (4). Considering the crucial roles of these metals, development of novel Fe and Cu chelators has become a promising anticancer strategy (1, 5). The chelator, Triapine (3-aminopyridine2-carboxaldehyde thiosemicarbazone; Fig. 1), which inhibits tumor growth, has entered clinical trials (1). The di2-pyridylketone thiosemicarbazone (DpT) chelators possess far greater antitumor activity and selectivity than Triapine (6, 7)
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