Abstract
The intracellular distribution of a drug can cause significant variability in both activity and selectivity. Herein, we investigate the mechanism by which the anti-cancer agents, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and the clinically trialed, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), re-instate the efficacy of doxorubicin (DOX), in drug-resistant P-glycoprotein (Pgp)-expressing cells. Both Dp44mT and DpC potently target and kill Pgp-expressing tumors, while DOX effectively kills non-Pgp-expressing cancers. Thus, the combination of these agents should be considered as an effective rationalized therapy for potently treating advanced and resistant tumors that are often heterogeneous in terms of Pgp-expression. These studies demonstrate that both Dp44mT and DpC are transported into lysosomes via Pgp transport activity, where they induce lysosomal-membrane permeabilization to release DOX trapped within lysosomes. This novel strategy of loading lysosomes with DOX, followed by permeabilization with Dp44mT or DpC, results in the relocalization of stored DOX from its lysosomal 'safe house' to its nuclear targets, markedly enhancing cellular toxicity against resistant tumor cells. Notably, the combination of Dp44mT or DpC with DOX showed a very high level of synergism in multiple Pgp-expressing cell types, for example, cervical, breast and colorectal cancer cells. These studies revealed that the level of drug synergy was proportional to Pgp activity. Interestingly, synergism was ablated by inhibiting Pgp using the pharmacological inhibitor, Elacridar, or by inhibiting Pgp-expression using Pgp-silencing, demonstrating the importance of Pgp in the synergistic interaction. Furthermore, lysosomal-membrane stabilization inhibited the relocalization of DOX from lysosomes to the nucleus upon combination with Dp44mT or DpC, preventing synergism. This latter observation demonstrated the importance of lysosomal-membrane permeabilization to the synergistic interaction between these agents. The synergistic and potent anti-tumor efficacy observed between DOX and thiosemicarbazones represents a promising treatment combination for advanced cancers, which are heterogeneous and composed of non-Pgp- and Pgp-expressing tumor cells.
Highlights
Doxorubicin (DOX; Figure 1ai), is resistance caused by drug-resistance pumps, for example, P-glycoprotein (Pgp).[1,2] This has been attributed to both Pgp-mediated drug efflux through the plasma membrane[3] and due to increased
DOX is stored in lysosomes due to it becoming protonated in the acidic pH of this organelle, preventing its distribution to its major targets in the nucleus, leading to drug resistance.[4]
A shorter 24 h/37 °C incubation was utilized for the highly cytotoxic thiosemicarbazones, Dp44mT and di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC),[5,6,7,8,9,10,11] as longer times led to almost total cellular ablation
Summary
Doxorubicin (DOX; Figure 1ai), is resistance caused by drug-resistance pumps, for example, P-glycoprotein (Pgp).[1,2] This has been attributed to both Pgp-mediated drug efflux through the plasma membrane[3] and due to increased. A novel class of anti-cancer agents, the di-2-pyridylketone thiosemicarbazone (DpT) analogs, has demonstrated potent and selective activity and the ability to overcome multidrug resistance by directly utilizing lysosomal Pgp-transport activity.[5,6] These agents include the first generation compound, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT; Figure 1bi),[7,8,9] and the more selective second generation analog, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC; Figure 1bii),[10,11] which has entered clinical trials (NCT02688101).[12] These agents are Pgp substrates and are effluxed out of cells, and transported into lysosomes by utilizing Pgp on the lysosomal membrane (Figure 1biii).[5,6,13] These agents bind copper and redox cycle to generate reactive oxygen species that induce lysosomalmembrane permeabilization (LMP), which results in apoptotic death, thereby overcoming Pgp-mediated resistance (Figure 1biii).[5,9,14,15,16] this mechanism is opposite to DOX (Figure 1aii), which does not induce lysosomal permeabilization and remains securely trapped within lysosomes, inducing resistance. Both these potent cytotoxic effects result in synergism between these agents that markedly
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