Comparison of three cytotoxicity tests in the evaluation of the cytotoxicity of a spermine analogue on human breast cancer cell lines

Using three cytotoxicity assays, we have investigated the effect of the spermine analogue N1,N11-diethylnorspermine (DENSPM) on four human breast cancer cell lines with different known genetic lesions. Cells were seeded in 96 well plates and DENSPM was added 24 h later to give final concentrations from 0.1 to 100 μM. At 24, 48 and 72 h of treatment, the protein content was determined with a modified Lowry assay. Mitochondrial activity was determined with the AlamarBlue™ and MTT assays. These two assays differ with respect to where in the electron transport chain the reduction of the substrate takes place. Treatment with increasing concentrations of DENSPM resulted in differential responses in the four cell lines. There was a good of agreement between the protein content and the MTT assay showing increased negative effect with increased dose of DENSPM. The AlamarBlue™ assay on the other hand showed a stimulation of substrate reduction compared to control at DENSPM concentrations that were inhibitory according to the protein content and MTT assay. Thus, the data clearly show that the MTT and AlamarBlue™ assays are not equivalent. Importantly, the AlamarBlue™ assay presumably also reflects cytoplasmic reduction of the substrate through DENSPM-induced mechanisms.