Aims:
This study investigated the mechanism by which the anti-cancer agents, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and the clinically trialled, 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.
Methods:
All cancer cell lines were assessed for P-gp protein expression and function using Western Blotting and Rhodamine 123 retention assays. Drug cytotoxicity was measured with MTT assays and drug synergy calculated using the Chou-Talalay method. DOX cellular localisation was visualised using fluorescence microscopy.
Results:
These studies demonstrated that both Dp44mT and DpC are transported into lysosomes via Pgp transport activity, where they induce lysosomal-membrane permeabilisation to release DOX trapped within lysosomes. This novel strategy of loading lysosomes with DOX, followed by permeabilisation with Dp44mT or DpC, results in the relocalisation of stored DOX from its lysosomal 'safe house' to its nuclear targets, markedly enhancing cellular toxicity against resistant tumour cells. Notably, the combination of Dp44mT or DpC with DOX showed a very high level of synergism in multiple Pgp-expressing cell types, including cervical, breast and colorectal (CI: 0.13 ± 0.08 – 0.76 ± 0.10) 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 silencing Pgp (CI: 0.78 ± 0.10 - 0.91 ± 0.14), demonstrating the importance of Pgp in the synergistic interaction. Furthermore, lysosomal-membrane stabilization inhibited the relocalisation of DOX from lysosomes to the nucleus upon combination with Dp44mT or DpC (CI: 0.98 ± 0.07 - 1.08 ± 0.09), preventing synergism. This latter observation demonstrated the importance of lysosomal-membrane permeabilisation to the synergistic interaction between these agents.
Conclusions:
The synergistic anti-tumour 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 tumour cells.