Replacing glucose media with galactose to evaluate mitochondrial toxicity of imiquimod

Abstract

The off-label use of imiquimod (IQ) for hemangioma treatment has shown clinical benefits. We have previously reported a selective di- rect IQ-cytotoxic effect on transformed H5V endothelial cells (EC) (hemangioma model) vs normal 1G11 EC. We observed a severe imbalance in antioxidant defense and apoptosis in H5V but not in 1G11. To further address this issue, we studied the possibility of IQ being a mitochondrial toxicant. H5V and 1G11 cells were treated with IQ (0-50 μg/mL) for 2, 4, 12 or 24 h and analyzed for reac- tive oxygen species (ROS) with DCFH2-DA probe and mitochondrial stress by MitoTrackerTM Red CMXRos fluorescence. Viability assays were performed using the standard culture medium with 5.5 mM glucose (regular) or media containing 25 mM glucose (high) or 25 mM galactose (depleted). IQ treatment increased ROS level in H5V after 2 h (35-60%; p<0.05) but in 1G11 only at 4 h (̴50%; p<0.05). Mitochondrial membrane potential in H5V cells was affected after 4 and 12 h treatment, revealed by a decreased in MitoTracker fluores- cence (≈50%; p<0.05). In contrast, 1G11 cells were unaffected and only presented a significant 30%-decrease in fluorescence after 12 h with 50 μg/mL IQ (p<0.05). Cells grown in a high glucose medium can adapt to a glycolytic phenotype. By assessing the effect of IQ in this medium, both cell lines became significantly less affected than with the regular culture medium. On the contrary, by forcing cells to respiration with galactose instead of glucose-containing medium, IQ treatment enhanced cell death in both cell lines, being fully cytotoxic for H5V (p<0.05) but leaving ≈32% 1G11 cells still alive at the high- est IQ concentrations.These results provide more evidences about the higher suscepti- bility of transformed EC to IQ, where an early ROS production and mitochondrial dysfunction drove H5V cells to death. By shifting cells towards diminished respiration in absence of glucose, we proved IQ acts as a mitochondrial toxicant in both EC lines.