Research On Curcumin Target Screening Based On Biotin Prob

Research backgroundCurcumin,derived from Curcuma longa,is a natural polyphenol compound,which has pharmacological effects such as anti-proliferation,anti-tumor,anti-inflammation,antioxidation,anti human virus,immune regulation,liver and kidney protection,and anti fibrosis.Biotin,as a natural biological label,has the potential to target and bind to tumor cells.Till now the target protein of curcumin has never been systematically studied.With the continuous development of Chemical genetics and chemical proteomics,it is feasible to determine the protein targets of a compound.Among them,biotinylation probe technology is widely used in target identification experiments.Research methodExperiment 1:CCK-8,cell immunofluorescence,cell scratch test,and apoptosis detection were used to explore whether the activity of biotin modified curcumin still exists.Experiment 2:(1)Cellular immunofluorescence:The localization of biotin modified curcumin in colorectal cancer HCT116 cells and liver cancer Hep3b cells was observed by using the tracer effect of fluorescent dyes.(2)Isolation of potential binding proteins:Biotin modified curcumin,Streptavidin binding amplification technology and polyacrylamide gel electrophoresis were used to separate potential binding proteins of curcumin in colorectal cancer HCT116 cells.(3)Mass spectrometry:Liquid phase mass spectrometry and tandem mass spectrometry were used to screen and isolate curcumin targeted proteins in colorectal cancer HCT116 cells,and biological functional analysis of the core proteins was carried out.(4)GO analysis:according to the mass spectrometry results,use the DAVID database to carry out GO enrichment analysis on potential curcumin binding proteins.(5)PPI network construction and IPA analysis:124 potential binding proteins of curcumin were imported into STRING database to construct a protein protein interaction(PPI)network,and integrated analysis was conducted using Ingenity Pathway Analysis(IPA)software,including signal pathways and metabolic pathways.(6)Molecular docking:10 proteins were screened for molecular docking according to the mass spectrum results,and the binding degree was analyzed.Research resultsExperiment 1:CCK-8,cellular immunofluorescence,cell scratch test and apoptosis detection results all confirmed that the activity of biotin modified curcumin still exists.Experiment 2:(1)Cell immune fluorescence:Compared with the control group(CTRL),the expression of green fluorescence in the biotin modified curcumin group was significantly enhanced.Besides,the green fluorescent dye can be observed to diffuse to the whole cell.We can see the general outline of the cell through the fluorescence area,and the fluorescence brightness of the nucleus is the strongest,and under the same condition.With the increase of the concentration of biotin modified curcumin,the fluorescence intensity also increased,indicating that more curcumin entered colorectal cancer HCT116 cells and liver cancer Hep3b cells.(2)Separation of potential binding proteins:The potential curcumin binding proteins in colorectal cancer HCT116 cells were separated by the combination amplification technology of biotin modified curcumin and Streptavidin and polyacrylamide gel electrophoresis.The results showed that the mobile phase was a non curcumin binding protein,the elution phase was a curcumin binding protein,and the molecular weight of potential curcumin binding protein was about 10-15 KDa.(3)Mass spectrometry:After liquid chromatography and tandem mass spectrometry analysis,124 potential binding proteins of curcumin were retrieved,some of which are closely related to oxidative stress and tumorigenesis.(4)GO analysis:Targets are widely distributed and enriched in the nucleus,mitochondria,and plasma membrane,participating in various biological functions such as metabolic processes,regulation,response to stimuli,and cellular processes.(5)PPI network construction and IPA analysis:curcumin may play a biological role through a variety of key biological pathways,including oxidative phosphorylation,EIF2,eIF4/p70S6K,mTOR signal transduction,mitochondrial dysfunction and other signal pathways and metabolic pathways.(6)Molecular docking results suggest that curcumin and key target proteins can form multiple covalent bond with good affinity.FABP1,PFN1 and H2B may be potential binding proteins of curcumin in colorectal cancer HCT116 cells.Research conclusion1 The biological activity of biotin modified curcumin still remains.2 124 potential binding proteins of curcumin were retrieved,and their main physiological functions were closely related to oxidative stress and tumorigenesis.The targets were widely distributed and enriched in the nucleus,mitochondria and plasma membrane,and involved in many biological functions such as metabolic process,regulation,response to stimulation and cell process.Curcumin may exert its biological functions be through oxidative phosphorylation,EIF2,eIF4/p70S6K,The mTOR signaling pathway and mitochondrial dysfunction play biological roles in signaling and metabolic pathways.3 FABP1,PFN1,H2B may be the potential binding of curcumin in colorectal cancer HCT116 cells.