Novel strategies to prevent the development of multidrug resistance (MDR) in cancer Novel strategies to prevent the development of multidrug resistance (MDR) in cancer

Cancer is currently the second leading cause of death globally and is expected to be responsible for approximately 9.6 million deaths in 2018. With an unprecedented understanding of the molecular pathways that drive the development and progression of human cancers, novel targeted therapies have become an exciting new development for anti-cancer medicine. These targeted therapies, also known as biologic therapies, have become a major modality of medical treatment, by acting to block the growth of cancer cells by specifically targeting molecules required for cell growth and tumorigenesis. Due to their specificity, these new therapies are expected to have better efficacy and limited adverse side effects when compared with other treatment options, including hormonal and cytotoxic therapies. In this review, we explore the clinical development, successes and challenges facing targeted anti-cancer therapies, including both small molecule inhibitors and antibody targeted therapies. Herein, we introduce targeted therapies to epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), human epidermal growth factor receptor 2 (HER2), anaplastic lymphoma kinase (ALK), BRAF, and the inhibitors of the T-cell mediated immune response, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein-1 (PD-1)/ PD-1 ligand (PD-1 L).

Multidrug-resistance (MDR) is a major obstacle in cancer treatment. More than half of human cancers express multidrug-resistant (MDR) P-glycoprotein (Pgp), which correlates with a poor prognosis. Intriguingly, through an unknown mechanism, some drugs have greater activity in drug-resistant tumor cells than their drug-sensitive counterparts. Herein, we investigate how the novel anti-tumor agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), overcomes MDR. Four different cell-types were utilized to evaluate the effect of Pgp-potentiated lysosomal targeting of drugs to overcome MDR. To assess the mechanism of how Dp44mT overcomes drug resistance, cellular studies utilized: Pgp inhibitors, Pgp-silencing, lysosomotropic agents, proliferation assays, immunoblotting, a Pgp-ATPase activity assay, radio-labeled drug uptake/efflux, a Rh123-retention assay, lysosomal-membrane permeability assessment and DCF redox studies. Anti-tumor activity and selectivity of Dp44mT in Pgp-expressing, MDR cells versus drug-sensitive cells were studied using a BALB/c nu/nu xenograft mouse model. We demonstrate that Dp44mT is transported by the lysosomal Pgp drug pump, causing lysosomal-targeting of Dp44mT and resulting in enhanced cytotoxicity in MDR cells. Lysosomal Pgp and pH were shown to be crucial for increasing Dp44mT-mediated lysosomal damage and subsequent cytotoxicity in drug-resistant cells, with Dp44mT being demonstrated to be a Pgp substrate. Indeed, Pgp-dependent lysosomal damage and cytotoxicity of Dp44mT were abrogated by Pgp inhibitors, Pgp-silencing, or by increasing lysosomal pH using lysosomotropic bases. In vivo, Dp44mT potently targeted chemotherapeutic resistant human Pgp-expressing xenografted tumors relative to non-Pgp-expressing tumors in mice. This study highlights a novel Pgp-hijacking strategy of the unique DpT series of thiosemicarbazones that overcome MDR via utilization of lysosomal-Pgp transport activity.

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.