HighlightsH/R induces autophagy and Nrf2 in tumoral and primary trophoblast cells.Melatonin inhibits autophagy and Nrf2 under H/R, inducing BeWo cell death.Melatonin increases autophagy and Nrf2 under H/R conditions, promoting primary villous trophoblast cells survival.AbstractHypoxia/reoxygenation (H/R) induces oxidative damage and apoptosis. These consequences activate autophagy, which degrades damaged cellular content, as well as inducing the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) transcription factor, and thereby the expression of protective genes. Melatonin has protective roles in normal cells and cytotoxic actions in cancer cells, with effects involving autophagy and Nrf2 pathways. The current study shows melatonin to differentially modulate autophagy and Nrf2 pathways in tumor and normal placental cells exposed to H/R. BeWo, a human placental choriocarcinoma cell line, and primary villous cytotrophoblasts isolated from normal term placenta, were maintained in normoxia (8% O2) for 24 h or exposed to hypoxia (0.5% of O2 for 4 h) followed by 20 h of normoxia, creating a H/R, in the presence or absence of 1 mM melatonin. Melatonin induced a 7-fold increase in the activation of 5’ adenosine monophosphate-activated protein kinase (AMPK)α, an upstream modulator of autophagy, rising to a 16-fold increase in cells co-exposed to H/R and melatonin, compared to controls. H/R induced autophagosome formation via the increased expression of Beclin-1 (by 94 %) and ATG7 (by 97%). H/R also induced autophagic activity, indicated by the by the 630% increase in P62, and increased Nrf2 by 314%. In H/R conditions, melatonin reduced autophagy by 74% and Nrf2 expression by 66%, leading to BeWo cell apoptosis. In contrast, in human primary villous cytotrophoblasts, H/R induced autophagy and Nrf2, which melatonin further potentiated, thereby affording protection against H/R. This study demonstrates that melatonin differentially modulates autophagy and the Nrf2 pathway in normal vs. tumor trophoblast cells, being cytoprotective in normal cells whilst increasing apoptosis in tumoral trophoblast cells.