Supplementary Material for: Effect of Arsenic Trioxide on Uveal Melanoma Cell Proliferation in vitro

Aims: The aim of this study was to investigate the effect of arsenic trioxide (ATO) on the growth of cultured uveal melanoma cells (OCM-1 cell line). Methods: Cultured OCM-1 cells were treated with 0.75–24 µM of ATO for 4–96 h. Cell viability was evaluated with the methylthiazoletetrazolium (MTT) assay as proliferation test. Apoptotic and necrotic cells were quantified using flow cytometry following Annexin-V/PI double stain. The cell morphology alteration was examined by light and electron transmission microscopy. To investigate the underlying mechanism of ATO-induced apoptosis and necrosis, glutathione peroxidase activity was measured, and mitochondrial membrane potentials were quantified using confocal microscopy. Results: In the MTT assays, OCM-1 cell growth was inhibited at ATO concentrations of 1.5–24 µM. The 50% inhibitory concentrationof ATO was 16.8 µM. At ATO concentrations of 12 and 24 µM, apoptosis and necrosis were induced after 24 h of incubation as shown by light and transmission electron microscopy. At ATO concentrations of 12 and 24 µM, the glutathione peroxidase activity was significantly (p < 0.05) reduced after 24 h of incubation, and the mitochondrial membrane potentials were significantly (p < 0.01) decreased after 4, 12 and 24 h of incubation. Conclusion: ATO inhibited ocular melanoma cell line growth at concentrations of 1.5–24 µM of ATO in a dose- and time-dependent manner by inducing apoptosis and necrosis of the tumor cells. The concurringly decreased glutathione peroxidase activity and the reduced mitochondrial membrane potentials may be possible underlying mechanisms for the apoptosis and necrosis of the tumor cells.