(E)-3-(4-chlorophenyl)-N-(7-hydroxy-6-methoxy-2-oxo-2H-chromen-3-yl) acrylamide (SC-III3), a newly synthesized derivative of
scopoletin, was previously shown to reduce the viability of HepG2 cells and
tumor growth of HepG2 xenograft mouse model. It induces the death of HepG2 cells by a way irrelevant to apoptosis and
necrosis. To shed light on the cytotoxic mechanisms of
SC-III3, the present study addresses whether and how it can induce autophagic cell death. When HepG2 cells were incubated with various concentrations of
SC-III3, autophagic vacuoles could be observed by transmission electron microscopy and
monodansylcadaverine staining. Increased expressions of LC3-II to LC3-I and
Beclin-1, required for autophagosome formation, were accompanied. These characteristics integrally indicated that
SC-III3 could initiate autophagy in HepG2 cells.
N-acetyl-l-cysteine (NAC), a ROS scavenger, could reverse SC-III3-caused ROS accumulation, but it did not affect SC-III3-induced autophagy, suggesting that ROS was not involved in SC-III3-mediated autophagy in HepG2 cells.
SC-III3 significantly depressed mitochondrial function, as evidenced by disruption of mitochondrial transmembrane potential and loss of the mitochondrial cristae structure, as well as decrease of Cox-I, Cox-III, Cox-IV, and
ATP levels. The autophagy and activation of AMPK-TSC2-mTOR-p70s6k pathways induced by
SC-III3 in HepG2 cells could be efficiently blocked by pre-treatments of compound C (an inhibitor of AMPK). Moreover, addition of extracellular
ATP to the cell
culture media could reverse SC-III3-caused activation of AMPK-TSC2-mTOR-p70s6k pathway, autophagy and cell viability decrease in HepG2 cells. Collectively,
SC-III3 leads to autophagy through inducing
mitochondrial dysfunction, depleting
ATP, and activating AMPK-mTOR pathway, which thus reflects the cytotoxic effect of
SC-III3 in HepG2 cells.