4-Amino-2-Trifluoromethyl-Phenyl Retinate Induced G₀/G₁ Phase Arrest In Gastric Cancer Cells By Regulating The Binding Of 14-3-3? And Filamin A

Gastric cancer is the fifth most common cancer in the world and the morbidity of this disease is very high in china,which also has received public concern as a health problem.However,because of the low rate of early diagnosis,most of the patients are treated in the middle and even late stages,so there is no hope of cure.The chemotherapy is the main treatment,but the outcome is often far from optimism due to the shortage of therapeutical-associated toxicity.The new treatment strategy should be developed so as to overcome the current problems.The principle of induced differentiation therapy is to induce the malignant tumor cells to evolve and differentiate in the direction of healthy cells so as to achieve the purpose of treating tumors and is an ideal target for future treatment of cancer.All-trans retinoic acid?ATRA?is the main drug in clinical for the treatment of acute promyelocytic leukemia?APL?.With the widespread use in clinical trials,the main side effects and adverse reactions of ATRA affected its application in the clinical setting.Based on the structural modification of ATRA,we had screened 4-amino-2-trifluoromethyl-phenyl retinate?ATPR?from the series derivatives and found it could induce the cell cycle arrest and differentiation effects for a variety of solid tumors.One of the potential mechanism of ATPR-induced SGC-7901 cells G₀/G₁ phase arrest and differentiation may be to inhibit the phosphorylation of Akt and down-regualte 14-3-3?expression.The 14-3-3 proteins,which belong in the molecular chaperones group,are involved in the regulation of multiple cell cycle signaling pathways in cells.ATPR can down-regulate the expression of 14-3-3?in our primary study,but the mechanism by which 14-3-3?affects the cell cycle related proteins to play its cell cycle arrest effect are our present research contents.ObjectiveTo screen the cell cycle related proteins from the 14-3-3?-binding proteins by use of immunoprecipitation,identification and bioinformatics analysis,so as to elucidate the ATPR-induced cell cycle arrest mechanism by regulating 14-3-3?and its binding proteins.MethodsSGC-7901 cells were incubated with vehicle?0.5%alcohol?and ATPR at the concentration of 1 x10^-55 mol/L for 48 hours and then total protein,cytoplasm protein and nuclear protein were extracted.?1?The isolation of 14-3-3?-binding proteins.Immunoprecipitated with anti-14-3-3?antibody,the extractives were subjected to vertical electrophoresis of SDS-PAGE and then the gel was stained with Coomassie Brilliant Blue dye.?2?The identification of 14-3-3?-binding proteins.The protein bands were excised from the one-dimensional Coomassie blue-stained polyacrylamide gel.The bands were digested in the gel with trypsin and the resulting tryptic peptides were then desalted and subjected to mass spectrometry analysis.The raw data files were analyzed using the SEQUEST search engine and the Proteome Discoverer software.By using the human non-redundant peptide database,the 14-3-3?-binding protein profits were obtained.?3?The bioinformatics analysis of 14-3-3?-binding proteins.The Uniprot accessions of identified proteins were uploaded on PANTHER classification systems.The proteins were annotated based on their biological processes,molecular functions and protein classes.The cell cycle related proteins among of them were screened out.?4?The validation of 14-3-3?-binding proteins.Double-direction immunoprecipitation combined with Western blot analysis were used to validate the binding of 14-3-3?and filamin A.In addition,the double immunofluorescent staining method was used observe the binding of 14-3-3?and filamin A.?5?The differential expressions of 14-3-3?and filamin A were analyzed by label-free quantitative proteomics analysis.The data collected from LC-MS/MS were imported into label-free quantitative proteomics software.The parameters setting at the level of expression for more than 1.5 folds,P Value<0.05 and at least 10%sequence coverage in peptide with above score 16,peptides matched were 3 or more.?6?After siRNA transfection to filamin A or 14-3-3?in SGC-7901,the effects of filamin A and ATPR treatment were analyzed on a flow cytometer.?7?Overexpression of 14-3-3?in SGC-7901 cells and then treated with ATPR for 48h.Total protein,nuclear and cytoplasmic proteins were extracted.Western blot method was used to analyze the expressions of filamin A and 14-3-3?in different subcellular partitions.?8?Immunohistochemistry assay was used to analyze the expressions of filamin A and14-3-3?in paired human gastric cancer samples?GC?and their adjacent non-tumorous gastric tissues?NT?.Results?1?The results of proteomics analysis showed that 352 proteins were deduced to be bound with 14-3-3?in the vehicle group and the ATPR group after redundant proteins were excluded.Among of them,35 proteins were identified as cell cycle related or differentiation related proteins by annotated with GO terms using PANTHER.Four proteins,including filamin A,SFN,Catenin beta-1,and ANXA1,were played functions simultaneously in cell cycle and in differentiation.?2?The binding of 14-3-3?and filamin A was validated by Double-direction immunoprecipitation combined with Western blot analysis,as well as the double immunofluorescent staining method.Meanwhile,compared with the Vehicle group,the expression of filamin A and the binding amount with 14-3-3?were decreased in the ATPR group.?3?The cell cycle was arrested at the G₀/G₁ phase when it was ATPR-induced or when filamin A was silenced.Notably,compared with the siRNA-filamin A group,the G₀/G₁phase distribution was not observably changed after ATPR treatment on siRNA-filamin A cells.?4?ATPR may regulate the subcellular localization of filamin A through 14-3-3?.Subcellular expression analysis showed that filamin A was transferred from the cytoplasm to the nucleus after ATPR treatment.When 14-3-3?was overexpressed,the expression of filamin A was increased.The subcellular distribution of filamin A was changed and increased in the cytoplasm.?5?The expression of 14-3-3?and filamin A were increased in gastric cancer tissues.Filamin A was mainly distributed in the nucleus in the adjacent normal tissues.However,there was no such trend in the gastric cancer tissues.ConclusionsOur study analyzed the biological processes,protein classes and molecular functions of 14-3-3?-binding proteins in gastric cancer cells before and after ATPR treatment.Furthermore,we found that the binding of 14-3-3?and filamin A was decreased after ATPR treatment.ATPR can regulate the subcellular localization of filamin A by reducing the binding of 14-3-3?and filamin A,which may be the mechanism of ATPR-induced cell cycle arrest.Finally,targeting 14-3-3?and its binding proteins might be an optional strategy for cancer treatment.