Ceramide metabolism regulates autophagy and apoptotic cell death induced by melatonin in liver cancer cells

<jats:title>Abstract</jats:title><jats:p>Autophagy is a process that maintains homeostasis during stress, although it also contributes to cell death under specific contexts. Ceramides have emerged as important effectors in the regulation of autophagy, mediating the crosstalk with apoptosis. Melatonin induces apoptosis of cancer cells; however, its role in autophagy and ceramide metabolism has yet to be clearly elucidated. This study was aimed to evaluate the effect of melatonin administration on autophagy and ceramide metabolism and its possible link with melatonin‐induced apoptotic cell death in hepatocarcinoma (<jats:styled-content style="fixed-case">HCC</jats:styled-content>) cells. Melatonin (2 m<jats:sc>m</jats:sc>) transiently induced autophagy in HepG2 cells through <jats:styled-content style="fixed-case">JNK</jats:styled-content> phosphorylation, characterized by increased Beclin‐1 expression, p62 degradation, and <jats:styled-content style="fixed-case">LC</jats:styled-content>3<jats:styled-content style="fixed-case">II</jats:styled-content> and <jats:styled-content style="fixed-case">LAMP</jats:styled-content>‐2 colocalization, which translated in decreased cell viability. Moreover, <jats:italic><jats:styled-content style="fixed-case">ATG</jats:styled-content>5</jats:italic> silencing sensitized HepG2 cells to melatonin‐induced apoptosis, suggesting a dual role of autophagy in cell death. Melatonin enhanced ceramide levels through both de novo synthesis and acid sphingomyelinase (<jats:styled-content style="fixed-case">ASM</jats:styled-content>ase) stimulation. Serine palmitoyltransferase (<jats:styled-content style="fixed-case">SPT</jats:styled-content>) inhibition with myriocin prevented melatonin‐induced autophagy and <jats:styled-content style="fixed-case">ASM</jats:styled-content>ase inhibition with imipramine‐impaired autophagy flux. However, <jats:styled-content style="fixed-case">ASM</jats:styled-content>ase inhibition partially protected HepG2 cells against melatonin, while <jats:styled-content style="fixed-case">SPT</jats:styled-content> inhibition significantly enhanced cell death. Findings suggest a crosstalk between <jats:styled-content style="fixed-case">SPT</jats:styled-content>‐mediated ceramide generation and autophagy in protecting against melatonin, while specific <jats:styled-content style="fixed-case">ASM</jats:styled-content>ase‐induced ceramide production participates in melatonin‐mediated cell death. Thus, dual blocking of <jats:styled-content style="fixed-case">SPT</jats:styled-content> and autophagy emerges as a potential strategy to potentiate the apoptotic effects of melatonin in liver cancer cells.</jats:p>