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Melatonin is a natural compound synthesized by a variety of organs. It has been described to possess cell protecting activity in normal cells but was shown to induce apoptotic cell death in cancer cells. We determined to which extent and based on which molecular mechanisms melatonin is able to cause apoptosis in HT-29 human colon cancer cells. Induction of apoptosis was assessed by caspase-3-like activity, nuclear fragmentation and chromatin condensation. Melatonin, when given alone at a concentration of 1 mM, did not affect any of the apoptosis markers. It potentiated apoptosis induced by the flavonoid flavone significantly. Whereas flavone alone at a concentration of 150 μM led to a 8-fold increase in caspase-3-like activity associated with around 40% of cells displaying DNA-fragmentation, a combination of flavone and melatonin increased caspase-3-like activity 30-fold and 80% of cells exhibited fragmentation of DNA when compared to untreated controls. Melatonin caused an increase in cytosolic lactate levels that most likely allows the flavone-induced activation of the mitochondrial pyruvate/lactate importer to deliver more substrates to mitochondrial respiration. The subsequent increased production of mitochondrial O2− · in the presence of flavone was further increased by melatonin. Scavenging mitochondrial O2−. by benzoquinone or blocking the lactate/pyruvate transporter by 5-nitro-2-(3-phenylpropylamino) benzoate inhibited mitochondrial O2−.-generation and apoptosis execution mediated by flavone and melatonin. Our study provides evidence that melatonin potentiates flavone-induced apoptosis in HT-29 human colon cancer cells by enhancing the level of oxidizable substrates that can be transported into mitochondria in the presence of flavone.