Optimization of the MTT Assay for B16 Murine Melanoma Cells and its Application in Assessing Growth Inhibition by Polyamines and Novel Polyamine Conjugates

Abstract

As part of our continuing development of new cytotoxins with potential anticancer activity, we have synthesized polyamine conjugates containing both the linear tetra-amine spermine and an acridine unit. Studies of growth inhibition by these novel conjugates and spermine were carried out using B16 murine melanoma cells. The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) bioassay has been optimized for this cell line with respect to incubation time for MTT metabolism, inoculum density and incubation period. A linear relationship has been established between the amount of formazan produced and the number of viable B16 murine melanoma cells. Furthermore, the optimal incubation time of cells with MTT is 3 h. Beyond this time no further significant amount of formazan was generated. The optimal seeding density was determined (4000 cells/well for 2- or 3- day experiments), and we estimate that the doubling time for B16 cells is approximately 24 h. In 48-h assays, spermine (1) inhibits cell growth with an EC50 of 450 μM, acylated analogue (5) has an EC50 of 5 μM, almost a hundred-fold increase in potency over spermine, and the aniline analogue (6) has an EC50 of 1 μM, a five-fold increase in potency over amide analogue (5). The EC50 for spermine (1) and aniline (6) changed little over 3 and 6 days compared with the EC50 over 2 days. We conclude that there is little significant metabolic influence (with time) on the observed toxicity data. We have also shown that the synthetic conjugates combining both an intercalator and groove binder show greater cytotoxicity than compounds that exhibit just one of these modes of binding. These analogues therefore offer a new lead in the design of cytotoxic polyamines with potential anticancer activity.