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Polyamines are small organic cations that are essential for many biological processes such as cell proliferation and cell cycle progression. While the metabolism of polyamines has been well studied, the mechanisms by which polyamines are transported into and out of cells are poorly understood. Here, we describe a novel role of Chmp1, a vesicular trafficking protein, in the transport of polyamines using a well-defined leg imaginal disc assay in Drosophila melanogaster larvae. We show that Chmp1 overexpression had no effect on leg development in Drosophila, but does attenuate the negative impact on leg development of Ant44, a cytotoxic drug known to enter cells through the polyamine transport system (PTS), suggesting that the overexpression of Chmp1 downregulated the PTS. Moreover, we showed that the addition of spermine did not rescue the leg development in Chmp1-overexpressing leg discs treated with difluoromethylornithine (DFMO), an inhibitor of polyamine metabolism, while putrescine and spermidine did, suggesting that there may be unique mechanisms of import for individual polyamines. Thus, our data provide novel insight into the underlying mechanisms that are involved in polyamine transport and highlight the utility of the Drosophila imaginal disc assay as a fast and easy way to study potential players involved in the PTS. [ABSTRACT FROM AUTHOR]

Polyamines are small polycationic alkylamines involved in many fundamental cellular processes, including cell proliferation, survival, and protection from oxidative stress. Polyamine homeostasis is tightly regulated through coordinated biosynthesis, catabolism, and transport. Due to their continual proliferation, cancer cells maintain elevated intracellular polyamine pools. Both polyamine metabolism and transport are commonly dysregulated in cancer, and as such, polyamine analogues are a promising strategy for exploiting the increased polyamine requirement of cancer cells. One potential polyamine analogue resistance mechanism is the downregulation of the poorly defined polyamine transport system. Recent advances in nanomedicine have produced nanostructures with polyamine analogue-based backbones (nanopolyamines). Similar nanostructures with non-polyamine backbones have been shown to be transported by endocytosis. As these polyamine-based nanoparticles could be a method for polyamine analogue delivery that bypasses polyamine transport, we designed the current studies to determine the efficacy of polyamine-based nanoparticles in cells lacking intact polyamine transport. Utilizing polyamine transport-deficient derivatives of lung adenocarcinoma lines, we demonstrated that cells unable to transport natural polyamines were also resistant to nanopolyamine-induced cytotoxicity. This resistance was a result of transport-deficient cells being incapable of importing and accumulating nanopolyamines. Pharmacological modulation of polyamine transport confirmed these results in polyamine transport competent cells. These studies provide additional insight into the polyamine transport pathway and suggest that receptor-mediated endocytosis is a likely mechanism of transport for higher-order polyamines, polyamine analogues and the nanopolyamines. [ABSTRACT FROM AUTHOR]

Pancreatic cancers are currently the fourth leading cause of cancer-related death and new therapies are desperately needed. The most common pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). This report describes the development of therapies, which effectively deplete PDAC cells of their required polyamine growth factors. Of all human tissues, the pancreas has the highest level of the native polyamine spermidine. To sustain their high growth rates, PDACs have altered polyamine metabolism, which is reflected in their high intracellular polyamine levels and their upregulated import of exogenous polyamines. To understand how these cancers respond to interventions that target their specific polyamine pools, L3.6pl human pancreatic cancer cells were challenged with specific inhibitors of polyamine biosynthesis. We found that pancreatic cell lines have excess polyamine pools, which they rebalance to address deficiencies induced by inhibitors of specific steps in polyamine biosynthesis (e.g., ornithine decarboxylase (ODC), spermidine synthase (SRM), and spermine synthase (SMS)). We also discovered that combination therapies targeting ODC, SMS, and polyamine import were the most effective in reducing intracellular polyamine pools and reducing PDAC cell growth. A combination therapy containing difluoromethylornithine (DFMO, an ODC inhibitor) and a polyamine transport inhibitor (PTI) were shown to significantly deplete intracellular polyamine pools. The additional presence of an SMS inhibitor as low as 100 nM was sufficient to further potentiate the DFMO + PTI treatment [ABSTRACT FROM AUTHOR]

Increased polyamine biosynthesis activity and an active polyamine transport system are characteristics of many cancer cell lines and polyamine depletion has been shown to be a viable anticancer strategy. Polyamine levels can be depleted by difluoromethylornithine (DFMO), an inhibitor of the key polyamine biosynthesis enzyme ornithine decarboxylase (ODC). However, malignant cells frequently circumvent DFMO therapy by up-regulating polyamine import. Therefore, there is a need to develop compounds that inhibit polyamine transport. Collectively, DFMO and a polyamine transport inhibitor (PTI) provide the basis for a combination therapy leading to effective intracellular polyamine depletion. We have previously shown that the pattern of uptake of a series of polyamine analogues in a Drosophila model epithelium shares many characteristics with mammalian cells, indicating a high degree of similarity between the mammalian and Drosophila polyamine transport systems. In this report, we focused on the utility of the Drosophila epithelial model to identify and characterize polyamine transport inhibitors. We show that a previously identified inhibitor of transport in mammalian cells has a similar activity profile in Drosophila. The Drosophila model was also used to evaluate two additional transport inhibitors. We further demonstrate that a cocktail of polyamine transport inhibitors is more effective than individual inhibitors, suggesting the existence of multiple transport systems in Drosophila. Our findings reinforce the similarity between the Drosophila and mammalian transport systems and the value of the Drosophila model to provide inexpensive early screening of molecules targeting the transport system. [ABSTRACT FROM AUTHOR]