Thiosemicarbazones Target Multiple Resistance Pathways in Cancer

Abstract

Multidrug resistance (MDR) is a significant obstacle in the successful treatment of cancer. To date, no therapies that target MDR have reached the clinic. Our thiosemicarbazone compound, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), induces greater cytotoxicity in drug resistant cancer cells that express the drug efflux transporter, P-glycoprotein (Pgp), than in their non-drug resistant counterparts. Herein, the structure-activity relationships of selected thiosemicarbazones were explored and the novel mechanism underlying their ability to overcome MDR was examined. Only thiosemicarbazones with electron-withdrawing substituents at the imine carbon mediated Pgp-dependent potentiated cytotoxicity and caused Pgp-dependent lysosomal membrane permeabilisation (LMP). This LMP relied on copper(II) chelation, reactive oxygen species generation and increased relative lipophilicity. We also synthesised fluorescent zinc(II) complexes of our potently anti-cancer thiosemicarbazones to assess their intracellular distribution. The Zn(II) complexes generally showed significantly greater cytotoxicity than the thiosemicarbazones alone. Confocal fluorescence imaging showed that the Zn(II) complex of our lead compound, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), was localised to lysosomes. Under lysosomal conditions, the Zn(II) complexes were shown to transmetallate with copper ions, leading to redox-active Cu(II) complexes that induced LMP and cytotoxicity. Furthermore, we investigated the anti-cancer efficacy of Dp44mT and DpC in Bcl-2 over-expressing, melanoma cells. Herein, we demonstrated that DpC decreased Bcl-2 expression, while Dp44mT did not. Furthermore, we showed that the mechanism by which DpC and Dp44mT exerted their cytotoxicity in melanoma cells did not involve copper ion binding or ROS generation. DpC, but not Dp44mT, induced autophagosome formation and increased the accumulation of acidic vesicles (e.g., autolysosomes), regardless of Bcl-2 expression. This thesis significantly expands current knowledge regarding novel strategies of overcoming MDR, which can be implemented in the design of innovative therapeutics.