Mechanisms of trichostatin A inhibiting AGS proliferation and identification of lysine-acetylated proteins

Abstract

Aim: 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.

Conclusion: 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.

Keywords: ATP5O; Acetylation modification; Gastric cancer; Mass spectrometry; PKM2; Trichostatin A.

Figure 1

Proliferation of AGS cells exposed to different concentrations of trichostatin A for 72 h. A: AGS cells after treatment with 0 μmol/L trichostatin A (TSA); B and C: AGS cells were significantly reduced after exposed to 0.25 μmol/L TSA (B) and further reduced after treated with 0.5 μmol/L TSA (C).

Figure 2

Effect of different concentrations of trichostatin A on inhibition of AGS cell proliferation in the cell counting kit-8 experiment. The inhibition of AGS cells was gradually increased with increasing trichostatin A concentration 0, 0.015, 0.03, 0.06, 0.1, 0.25, 0.5 and 1 μmol/L. TSA: Trichostatin A.

Figure 3

AGS cell apoptosis changes before and after trichostatin A exposure by flow cytometry. Fluorescence staining was mainly seen in normal areas, but rarely in apoptotic and necrotic areas of unexposed AGS cells; staining appeared in the apoptotic and necrotic areas of AGS cells exposed to 0.25 μmol/L trichostatin A (TSA).

Figure 4

AGS cell cycle changes before and after trichostatin A exposure by flow cytometry. The AGS cell cycle ratio before TSA treatment was: %G1 = 26, %S = 53.5, %G2 = 17.7, and the AGS cell cycle ratio after 0.25 μmol/L trichostatin A treatment was: %G1 = 44.6, %S = 20.9, %G2 = 31.3. Cycle arrest occurred in G0/G1, G2/M phases, especially in G0/G1 phase. TSA: Trichostatin A.

Figure 5

mRNA expression levels of p21, p53, Bax, Bcl-2, CDK2 and CyclinD1 in AGS cells exposed to 0.25 μmol/L trichostatin A shown by real-time polymerase chain reaction. The mRNA expression levels of Bcl-2, CDK2 and CyclinD1 were decreased and the mRNA expression levels of p21, p53 and Bax were increased 12 h after AGS cells were exposed to 0.25 μmol/L trichostatin A. The mRNA expression levels of Bcl-2, CDK2 and CyclinD1 were further decreased and the mRNA expression levels of p21, p53 and Bax were further increased 24 h after exposure.

Figure 6

Protein expression levels of p21, p53, Bax, Bcl-2, CDK and cyclin after AGS cells were exposed to 0.25 μmol/L trichostatin A shown by Western blotting. The protein expression levels of Bcl-2, CDK2 and CyclinD1 were decreased and the protein expression levels of p21, p53 and Bax were increased 12 h after AGS cells were exposed to 0.25 μmol/L trichostatin A. The protein expression levels of Bcl-2, CDK2 and CyclinD1 were further decreased and the protein expression levels of p21, p53 and Bax were further increased 24 h after exposure.

Figure 7

Identification of differential proteins of lysine acetylation after trichostatin A treatment. A: Silver staining showed the differential proteins of lysine acetylation in AGS cells after trichostatin A (TSA) treatment; B: Western blotting showed the differential proteins of lysine acetylation in AGS cells after TSA treatment, “-” before TSA intervention; “+” after TSA intervention, Acetyl-α-tubulin (Lys40) (D20G3) XP^® Rabbit mAb was the primary antibody and Goat anti-rabbit IgG-HRP was the secondary antibody.

Figure 8

Identification of the effectiveness of lysine-acetylated antibodies enriching acetylated proteins. Line 1: 20 μg total protein from AGS cells unexposed to trichostatin A (TSA); Line 2: 20 μg total protein from AGS cells exposed to 0.5 μmol/L TSA; Line 3: 20 μg flow-through protein from AGS cells unexposed to TSA, which was incubated with an antibody gel column; Line 4: 20 μg flow-through protein from AGS cells exposed to 0.5 μmol/L TSA, which was incubated with an antibody gel column; Line 5: 100 ng enriched protein from AGS cells unexposed to TSA, which was incubated with an antibody gel column; and Line 6: 100 ng enriched protein from AGS cells exposed to 0.5 μmol/L TSA, which was incubated with an antibody gel column.

Figure 9

Mass spectrometry total ion chromatography. A: 72 kDa band of lysine-acetylated protein in AGS cells before trichostatin A (TSA) treatment; B: 28 kDa band of lysine-acetylated protein in AGS cells after TSA treatment.

Figure 10

Mass spectrometry identification of peptides from silver staining gel. Mass spectra of 2 acetylated peptides from PKM2 and ATP5O are presented. A: PKM2 “AEGSDVANAVLDGADCIMLSGETAK”; B: ATP5O “FSPLTTNLINLLAENGR”.

Figure 11

G0 contents based on biological process, cellular components and molecular function in which differently modified proteins were significantly enriched. A: Biological process; B: Cellular components; C: Molecular function.

Figure 12

High-resolution MS/MS spectra of acetylated peptide at lysine 158 (GEVPCTVTSASPLEEATLSELK*TVLK) in ATP5O.

Figure 13

AGS cells were exposed to 0.5 μmol/L trichostatin A for the indicated time periods and then ATP5O and PKM2 protein were immunoprecipitated using an anti-ATP5O antibody and an anti-PKM2 antibody. Total ATP5O and acetylation of ATP5O were detected using an anti-ATP5O antibody and an antibody specific to acetylated lysine, respectively. Total PKM2 and deacetylation of PKM2 were detected using an anti-PKM2 antibody and an antibody specific to deacetylated lysine, respectively. ATP5O, PKM2 and β-Actin protein levels in the total lysate are also shown.