Aim: 

We have evaluated the effect of melatonin on apoptosis in the human leukemia cell line HL-60.

Methods: 

Caspase-3 and -9 activities were determined from the cleavage of the respective specific fluorogenic substrates and measured using a spectrofluorophotometer. The permeability transition pore (PTP) induction was determined with calcein in the presence of cobalt chloride, which quenches calcein fluorescence from all cellular domains except within mitochondria. Apoptosis and/or necrosis, and the cell cycle phase distribution were analysed by flow cytometry using Annexin V-FITC and propidium iodide. The amount of proapoptotic protein activated was determined by immunoprecipitation and western blotting.

Results: 

1 mM melatonin induced a significant increase in caspase activities. The effect of melatonin was time dependent, reaching a maximal caspase activity after 12 h of stimulation, and then decreased reaching a minimum after 72 h. Cell exposure to melatonin resulted in activation of Bax, which was found to be similar to the time-dependent bimodal effect on caspase activities. Melatonin also evoked PTP induction, which caused a loss of mitochondrial staining by calcein, and increased cell death by necrosis as demonstrated by propidium iodide positive cells after 72 h of stimulation. Similar results have been obtained from cells that were pretreated with the well-known apoptotic agent staurosporine (1 mg/ml for 2 hours).

Conclusion: 

We conclude that melatonin has proapoptotic and/or oncostatic time-dependent effects on the human myeloid cell line HL-60.

Supported by MEC-DGI and Junta de Extremadura grants BFU2007-60091 and PRI07-A024, respectively. I. Bejarano was beneficiary of grant by Junta de Extremadura PRE06070 and PC Redondo was supported by MEC (RYC2007-00349).