title: 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.
authors.affiliation: Department of Infectious Disease, The First Affiliated Hospital of Zhengzhou University, 1 Jianshendong Road, Zhengzhou, 450000, China.
authors.forename: Ranran
authors.initials: R
authors.lastname: Sun
authors.forename: Yonggang
authors.initials: Y
authors.lastname: Luo
authors.forename: Juan
authors.initials: J
authors.lastname: Li
authors.forename: Qiongye
authors.initials: Q
authors.lastname: Wang
authors.forename: Jingjing
authors.initials: J
authors.lastname: Li
authors.forename: Xiaolong
authors.initials: X
authors.lastname: Chen
authors.forename: Kelei
authors.initials: K
authors.lastname: Guan
authors.forename: Zujiang
authors.initials: Z
authors.lastname: Yu
mesh_terms: D000643:Ammonium Chloride / Q000008:administration & dosage*; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins; D001343:Autophagy / Q000187:drug effects; D000071193:Autophagy-Related Protein 7; D000071186:Beclin-1; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D056945:Hep G2 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D008565:Membrane Proteins; D016601:RNA-Binding Proteins / Q000096:biosynthesis; D015398:Signal Transduction / Q000187:drug effects; D020123:Sirolimus / Q000008:administration & dosage; D051899:Smad2 Protein / Q000096:biosynthesis / Q000235:genetics*; D044764:Ubiquitin-Activating Enzymes / Q000096:biosynthesis
chemical_list: D051017:Apoptosis Regulatory Proteins; C491997:BECN1 protein, human; D000071186:Beclin-1; D008565:Membrane Proteins; C118585:P62 protein, human; D016601:RNA-Binding Proteins; C497782:SMAD2 protein, human; D051899:Smad2 Protein; D000643:Ammonium Chloride; C509349:ATG7 protein, human; D000071193:Autophagy-Related Protein 7; D044764:Ubiquitin-Activating Enzymes; D020123:Sirolimus