Abstract
Melatonin has protective roles in normal cells and cytotoxic actions in cancer cells, with effects involving autophagy and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) transcription factor pathways. Hypoxia/reoxygenation (H/R) induces oxidative damage and apoptosis. These consequences activate autophagy, which degrades damaged cellular content, as well as activates Nrf2 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 situation of 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 BeWo 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%) in BeWo cells. Moreover, H/R also induced autophagic activity, indicated by the by the 630% increase in P62, and increased Nrf2 by 314% in BeWo cells. In H/R conditions, melatonin reduced autophagic activity by 74% and Nrf2 expression activation by 300%, 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.
Highlights
Macroautophagy, referred to as autophagy, is a highly conserved detoxifying mechanism involving the catabolism of damaged proteins and organelles [1]
Cells exposed to H/R and rapamycin, which induces autophagy, showed no differences in cell viability compared to normoxia, suggesting that rapamycin may improve cell viability in BeWo cells exposed to the suboptimal oxygen
SQSTM1/P62 shares a positive feedback loop with the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) [5], with Nrf2 showing significantly higher protein levels in BeWo cells exposed to H/R compared to normoxia, with Nrf2 levels similar to that of cells exposed to the positive control, sulforaphane, an Nrf2 activator (Fig 4B)
Summary
Macroautophagy, referred to as autophagy, is a highly conserved detoxifying mechanism involving the catabolism of damaged proteins and organelles [1]. Autophagy shows low levels of activity under basal conditions, being inhibited by the cellular sensor, the mechanistic target of rapamycin (mTOR). Autophagy is activated in suboptimal conditions, such as hypoxia/reoxygenation (H//R) or amino acid starvation (reviewed in [2]). Beclin-1 is an important initiator of autophagy via its activation of the ATG (autophagy-related) proteins. ATG proteins build a double-membrane vesicle, autophagosome, which engulfs cargo to be degraded in lysosomes. The consequent release of simpler structures can restore cellular energy levels and inhibit the deleterious effects of reactive species of oxygen (ROS) [3, 4]. Nrf is activated in during hypoxia in both normal and cancer cells, including placental cells [6,7,8]
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