Ammonium chloride inhibits autophagy of hepatocellular carcinoma cells through SMAD2 signaling

Abstract

Autophagy is a cellular degradation process for the clearance of damaged or superfluous proteins and organelles, the recycling of which serves as an alternative energy source during periods of metabolic stress to maintain cell homeostasis and viability. The anti-necrotic function of autophagy is critical for tumorigenesis of many tumor cells, including hepatocellular carcinoma (HCC). However, the underlying mechanism is not clarified yet. Ammonium chloride (NH₄Cl) is a well-known autophagy inhibitor, whereas its interaction with SMAD2 signaling pathway has not been reported previously. Here, we show that NH₄Cl significantly inhibited rapamycin-induced autophagy in HCC cells through decreasing the levels of Beclin-1, autophagy-related protein 7 (ATG7), p62, and autophagosome marker LC3 and significantly decreased the level of phosphorylated SMAD2 in rapamycin-treated HCC cells. In order to find out whether NH₄Cl may inhibit the autophagy in rapamycin-treated HCC cells through inhibition of SMAD2 signaling, we used transforming growth factor β1 (TGFβ1) to induce phosphorylation of SMAD2 in HCC cells. We found that induction of SMAD2 in HCC cells completely abolished the inhibitory effect of NH₄Cl on rapamycin-induced autophagy in HCC cells, suggesting that NH₄Cl inhibits autophagy of HCC cells through inhibiting SMAD2 signaling.