Investigation The Phosphoproteomics Of The Differentiation Of A Derivative Of All Trans Retinoic Acid-4-Amino-2-Trifluoromethyl-Phenyl Retinate On Human Gastric Cancer SGC-7901 Cells

As a commonly used differentiation inducing agents, All-trans retinoic acid(ATRA) has been applied to the treatment of acute promyelocytic leukemia, the mechanism is that inducing tumor cancer cells into normal cells, or inducing apoptosis. Clinical application found that retinoic acid drugs have defects including serious retinoic acid syndrome, drug-resistance and high recurrence rate, which induced the restriction of ATRA application in the clinical. 4-Amino-2-Trifluoromethyl-Phenyl Retinate(ATPR) is one of the retinoid derivatives designed and synthesized in our team, and its anti-proliferation and differentiation induction effects have been confirmed in our previous studies, which was expected to become a novel antineoplastic drugs. We choose human gastric cancer SGC-7901 cells as the research object to study different expression of protein when ATPR on gastric cancer cells, and find the targets of differentiation induction to explain that mechanism by phosphoproteomics.Objective: To investigate levels of phosphorylation of protein when the new retinoid derivatives 4-Amino-2-Trifluoromethyl-Phenyl Retinate(ATPR) on gastric cancer SGC-7901 cells, which can help to understand the possible mechanism of differentiation,and find the targets of differentiation induction to explain that mechanism by phosphoproteomics.Methods:Experiments are divided into two groups, ATPR group(final concentration 10-6mol/L) and control group, Gastric cancer SGC-7901 cells were incubated for 48 hours, collected and extracted of total cell protein, using the technique of phosphorproteomic to research, total cell proteins were digested by trypsin, phosphopeptides were enriched by Ti O2 beads and analyzed by LC-MS/MS. Use the Xcalibur 2.1 workstations and Proteome Discoverer1.2 software to found difference proteins and phosphosites compared with control group. Bioinformatic were applied to analyze the function and subcellular localization of phosphorylated proteins respectively to explain these different proteins by DAVID, KEGG, IPA software.Results:We identified 109 proteins, including 45 phosphoproteins and 121 phosphosites by analysis of MS, containing 82 serine phosphosites, 33 threonine phosphosites, 6 tyrosine phosphosites. Only 15 phosphoproteins appeared on the ATPR group compared with control group, 20 proteins only occured in the control group compared with control group. DAVID analysis results displayed that gastric cancer SGC7901 cell after treated with ATPR within phosphorproteins participated in regulating cellular process, and biological process, and biology, and gene expression, and macromolecules metabolic process, and primary metabolic process, and cellular metabolic process and so on; and according to subcellular localization analysis, different phosphoproteins mainly distributed in intracellular, and cytoplasmic and nucleus; The molecular functions of different phosphoproteins may including protein binding and nucleic acid binding. KEGG analysis results displayed that different phosphoproteins may involved ERBBsignaling pathway, RNA degradation, RNA transport, Protein processing in endoplasmic reticulum, Spliceosome, p53 signaling pathway. IPA software analyzed the relevance of the different phosphoproteins, discovered the ubiquitin ligase(UBC) became associated protein of between Calpastatin(CAST) and Adenylyl cyclase-associated protein 1(CAP1), Dna J homolog subfamily C member 5(DNAJC5) and Nuclear cap-binding protein subunit 1(NCBP1), Enhancer of m RNA-decapping protein 4(EDC4) and Tuberin(TSC2), suggested ubiquitin ligase may involved in regulating RNA and apoptotic processes. From the differential phosphoproteins, Hepatoma-derived growth factor(HDGF), CAST, ATP-binding cassette sub-family G member 4(ABCG4), and CAP1 might play an important role.Conclusion:1. 4-Amino-2-Trifluoromethyl-Phenyl Retinate(ATPR) regulated biological process, and gene expression, and metabolism process, and protein binding and nucleic acid binding. ERBB signaling pathway, RNA degradation, RNA transport, Protein processing in endoplasmic reticulum, spliceosome, p53 signaling pathway may involved, thus played role in induced human gastric cancer SGC-7901 cell differentiation.2.2.ATPR can induced differentiation of gastric cancer cell line SGC-7901, the mechanism may be the results of the interaction of multiple proteins or genes, such as HDGF, Calpastatin, ABCG4 and CAP1, and regulated the phosphorylation of these proteins, further research may confirm these proteins, so as to better explains the mechanism of ATPR.