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Effect of Macamides N‐(benzyl)linolenamide and N‐(3‐methoxybenzyl)linolenamide on Autophagy in HepG2 and HeLa Cells

Autophagy is a conserved catabolic process which channels intracellular proteins and organelles into lysosomes to be degraded. This process can be triggered either by nutrient deprivation or by metabolic stress. Autophagy malfunction has been linked to a variety of human diseases such as neurodegeneration, myopathies, infectious diseases and different cancer types. Therefore, the search for novel autophagy modulators is of great interest to reveal autophagic mechanisms involved in these disease processes. As many natural product‐derived compounds have been used as traditional medicines, they have great potential as lead autophagy modulators. Macamides are secondary metabolites isolated from the Peruvian plant Lepidium meyenii L. (Maca). These metabolites have exhibited various biological activities, including anti‐oxidative, anti‐fatigue, anti‐cancer, anti‐osteoporotic and neuroprotective effects. The purpose of this study was to evaluate the effect of two macamides: N‐(benzyl)linolenamide (Cmpd 1) and N‐(3‐methoxybenzyl)linolenamide (Cmpd 2) on autophagy in both HepG2 and HeLa cancer cell lines. Cell viability in both cell lines was strongly reduced by both Cmpd 1 (HepG2, IC50 = 75.6 μM; HeLa, IC50 = 116 μM) and Cmpd 2 (HepG2, IC50 = 62.8 μM; HeLa, IC50 = 117 μM) as determined by the MTS assay. Subsequently, it was investigated whether macamides inhibit lysosomal acidification, by monitoring the red fluorescence emitted by cells stained with LysoTracker Red DND‐99. Significantly, both Cmpd 1 and Cmpd 2 (50 μM) decreased the red fluorescence in both cell lines. Furthermore, to evaluate autophagic flux in HepG2 and HeLa cells, protein levels of microtubule‐associated protein 1A/1B‐light chain 3 (LC3) and p62 were measured. No significant differences were observed in LC3 levels. Nonetheless, Cmpd 1 was identified as the most effective compound to induce accumulation of p62 in HepG2 cells. Because p62 is degraded in the lysosome during the last stage of autophagy, these results suggest that macamides inhibit autolysosomal degradation. However, a more in‐depth study is needed to determine the inhibitory mechanism and/or if another type of autophagy is involved.