Melatonin enhances hydrogen peroxide-induced apoptosis in human dental pulp cells

BACKGROUND/PURPOSE: Melatonin, at physiological concentrations, was previously found to inhibit proliferation and promote odontogenic differentiation in human dental pulp cells (hDPCs), but its effect on apoptosis is unclear. Our study aimed to investigate the effect of melatonin on the H(2)O(2)-mediated viability reduction and apoptosis in hDPCs. MATERIALS AND METHODS: hDPCs were treated with H(2)O(2) (0, 250, 500, 1000 μmol/L), melatonin (0, 10(−12), 10(−10), 10(−8) mol/L), and melatonin with H(2)O(2) for 24 h. CCK-8 assays were performed to evaluate cell viability. Apoptosis was measured by DAPI and Annexin V/propidium iodide staining. Intracellular reactive oxygen species (ROS) were measured by CellROX® staining and mitochondrial membrane potential (ΔΨm) was examined by JC-1 staining. RESULTS: H(2)O(2) obviously decreased the viability of hDPCs in a concentration-dependent manner and melatonin alone also reduced viability by 16–20%. Melatonin was also found to enhance H(2)O(2)-induced toxicity in a concentration-dependent manner, and the highest physiological concentration of melatonin (10(−8) mol/L) had the most obvious effect (P < 0.001). Treating H(2)O(2)-exposed hDPCs with melatonin significantly increased the ratio of apoptotic cells with condensed and deformed nuclei (P < 0.001), as well as the percentage of Annexin V-positive cells (P < 0.01). Furthermore, melatonin significantly increased intracellular ROS levels and induced the loss of ΔΨm in H(2)O(2)-exposed cells (P < 0.05). CONCLUSION: Our results indicate that melatonin, at physiological concentrations, can enhance H(2)O(2)-induced apoptosis in hDPCs and increase H(2)O(2)-mediated ROS production and ΔΨm loss. Further studies are needed to investigate whether melatonin targets the mitochondrial death pathway during the process.