Polyamine transport inhibitors: Methods and caveats associated with measuring polyamine uptake in mammalian cells.

Combination therapies which target both polyamine biosynthesis and polyamine transport have shown promise as anti-cancer strategies and as potentiators of the immune response. While polyamine biosynthesis inhibitors like difluoromethylornithine (DFMO) exist, cancers often escape via upregulated polyamine import. As a result, polyamine transport inhibitors (PTIs) are needed to inhibit polyamine uptake and create a 'full-court press' on polyamine metabolism. As new PTIs are developed, they need to be ranked for their ability to inhibit polyamine uptake. This paper describes three polyamine transport assays to evaluate polyamine transport inhibition. The first tests the ability of the PTI to inhibit the uptake of an anthracene-containing polyamine poison (Ant44). The second assay evaluates the ability of the PTI to inhibit the uptake of a rescuing dose of spermidine into DFMO-treated cells. The final assay is the gold standard for the field and involves determining the concentration of PTI needed to inhibit 50 % of the uptake of each of the radiolabeled native polyamines: ³H-putrescine, ³H-spermidine or ¹⁴C-spermine. These assays provide EC₅₀ and IC₅₀ values which allow a formal ranking of transport inhibition potency to aid in PTI selection.