Quantitative Proteomics Study Of A Novel Ent-kaurene Diterpenoid YH-12

Flexicaulin A(here in after referred to as compound FA)is a new structure ent-kaurene diterpenoid isolated from Rabdosia rubescens by our group.Using it as the leading compound,our group synthesized several series of derivatives in the early stage.Compound YH-12 is a 14-position nicotinic acid esterified derivative of compound FA.It has high antitumor activity in vitro,so it has good research and development value.In this paper,quantitative proteomics was used to explore the anti-tumor mechanism of compound YH-12,and the conclusions were verified by biology.The specific contents are as follows:1.The anti-proliferative activity of compound YH-12 against eight common human cancer cell lines were determined by SRB method.The results showed that compound YH-12 has good inhibition on all of these cells,which has the strongest effect on Eca109 cells,exhibit IC50 values of 0.205 ?M and the inhibition is related to concentration and time.2.The quantitative proteomics of compound YH-12 was studied by TMT combined with nanoLC-MS/MS.The methods are shown as below:4?M compound YH-12 or DMSO was cultured with Eca109 cells for 36 hours respectively,and digested cell lysates were labeled with TMT 6 plex.The labeled samples were pre-fractionated into 24 fractions by high pH reversed phase chromatography.Instruments Easy-nLC 1000 combined with Q Exactive for nanoLC-MS/MS detection,MaxQuant searched the database.After statistical analysis,7431 proteins were identified,of which 6619 were quantifiable.From the 6619 quantifiable proteins,654 were found significant regulated(fold change? 1.3 or ?0.77,and p-value<0.05).Among them,333 up-regulated and 321 down-regulated.3.Gene Ontology and KEGG analysis and network pathway drawing were performed using DAVID website and Cytoscape software.The results are as follows:the functional pathways of compound YH-12 in tumor cells are mainly involved in metabolic pathway,N-glycan biosynthesis,lysosome,SNARE interactions in vesicular transport,valine,leucine and isoleucine degradation,fatty acid metabolism,peroxisome,protein export,fatty acid elongation,protein processing in endoplasmic reticulum,glycerolipid metabolism,ribosome,fanconi anemia pathway,cell cycle,p53 signal pathway,pyrimidine metabolism,purine metabolism and so on.4.Biological verification of the proteomics results were as follows:Cell cycle analysis showed that YH-12 induced G2/M phase arrest in Eca109 cells.Apoptosis analysis showed that YH-12 induced Eca109 to produce concentration-dependent apoptosis.The analysis of the KEGG pathway indicates that regulation of cell cycle by YH-12 involves the ATR-Chkl-Cdc25C pathway which related to G2/M phase,western blot showed that the expression levels of ATR,Chkl and Cdc25C protein were significantly down-regulated after treatment with YH-12.which was consistent with the results of proteomics data.In summary,the antitumor activity of compound YH-12 was tested in vitro.The antitumor mechanisms were studied by quantitative proteomics labeled with TMT and multiple targets and pathways were found.It was found for the first time by using proteomics that the anti-proliferative activity of these compounds was related to ATR,Chkl and Cdc25C proteins and verified by biology.The results of this paper can provide powerful clues and ideas for the further study of the anti-tumor mechanism and target discovery of this kind of compounds.