title: Mechanisms of trichostatin A inhibiting AGS proliferation and identification of lysine-acetylated proteins.
abstract: OBJECTIVE
  To explore the effect of lysine acetylation in related proteins on regulation of the proliferation of gastric cancer cells, and determine the lysine-acetylated proteins and the acetylated modified sites in AGS gastric cancer cells.
  
  
  METHODS
  The CCK-8 experiment and flow cytometry were used to observe the changes in proliferation and cycle of AGS cells treated with trichostatin A (TSA). Real time polymerase chain reaction and Western blotting were used to observe expression changes in p21, p53, Bax, Bcl-2, CDK2, and CyclinD1 in gastric cancer cells exposed to TSA. Cytoplasmic proteins in gastric cancer cells before and after TSA treatment were immunoprecipitated with anti-acetylated lysine antibodies, separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and silver-stained to detect the proteins by mass spectrometry after removal of the gel. The acetylated proteins in AGS cells were enriched with lysine-acetylated antibodies, and a high-resolution mass spectrometer was used to detect the acetylated proteins and modified sites.
  
  
  RESULTS
  TSA significantly inhibited AGS cell proliferation, and promoted cell apoptosis, leading to AGS cell cycle arrest in G0/G1 and G2/M phases, especially G0/G1 phase. p21, p53 and Bax gene expression levels in AGS cells were increased with TSA treatment duration; Bcl-2, CDK2, and CyclinD1 gene expression levels were decreased with TSA treatment duration. Two unknown protein bands, 72 kDa (before exposure to TSA) and 28 kDa (after exposure to TSA), were identified by silver-staining after immunoprecipitation of AGS cells with the lysine-acetylated monoclonal antibodies. Mass spectrometry showed that the 72 kDa protein band may be PKM2 and the 28 kDa protein band may be ATP5O. The acetylated proteins and modified sites in AGS cells were determined.
  
  
  CONCLUSIONS
  TSA can inhibit gastric cancer cell proliferation, which possibly activated signaling pathways in a variety of tumor-associated factors. ATP5O was obviously acetylated in AGS cells following TSA treatment.
authors.affiliation: Department of Gastroenterology, Shanghai Changning Central Hospital, Shanghai 200336, China.
authors.forename: Yu-Gang
authors.initials: YG
authors.lastname: Wang
authors.forename: Na
authors.initials: N
authors.lastname: Wang
authors.forename: Guang-Ming
authors.initials: GM
authors.lastname: Li
authors.forename: Wen-Li
authors.initials: WL
authors.lastname: Fang
authors.forename: Jue
authors.initials: J
authors.lastname: Wei
authors.forename: Jia-Li
authors.initials: JL
authors.lastname: Ma
authors.forename: Ting
authors.initials: T
authors.lastname: Wang
authors.forename: Min
authors.initials: M
authors.lastname: Shi
mesh_terms: D000107:Acetylation; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology*; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology*; D008239:Lysine; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D015398:Signal Transduction / Q000187:drug effects; D013274:Stomach Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D013997:Time Factors
chemical_list: D000970:Antineoplastic Agents; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D009363:Neoplasm Proteins; C012589:trichostatin A; D008239:Lysine
keywords: ATP5O; Acetylation modification; Gastric cancer; Mass spectrometry; PKM2; Trichostatin A