Research On Transmembrane Mechanism Of The Disulfide Bonds Of FITC-PI Targeting In Human Breast Cancer

Objectives:In our previous study,we found a 11-peptide with specific affinity for human three-negative breast cancer cells(MDA-MB-231)and human lung cancer cells(Calu-1).After FITC fluorescent labeling,we observed that it could penetrate into MDA-MB-231 and Calu-1 cells and locate in the cytoplasm.Further experiments showed that the 11-peptide added with disulfide ring can penetrate the cell membrane efficiently.The results indicated that the novel 11-peptide can be explored as a potential target carrier for cancer treatment.Based on the results of the previous studies,this paper studies the targeting transmembrane transfer mechanism of the disulfide bonds of FITC-PI,and explore the membrane proteins which help the disulfide bonds of FITC-PI penetrating into MDA-MB-231 and Calu-1 cells,moreover,we further investigate the cytoplasmic proteins which is associated with the process of transduction of the disulfide bonds of FITC-PI into the cells.Our work will directly related to the targeted therapy and early diagnosis of FITC-PI.Methods:1.20 STRs and amelogenin gene loci of human breast cancer MDA-MB-231 and human lung cancer Calu-1 cells were detected and mapped to the corresponding cell lines of ATCC,DSMZ,JCRB and RIKEN,after MDA-MB-231 and Calu-1 cells keeping in our laboratory checking without cross-contamination,and then the disulfide bonds of FITC-PI were synthesized.2.Four cell membrane nonspecific channel inhibitors(including chlorpromazine,nystatin,amiloride hydrochloride and chloroquine phosphate)were applied to block macropinocytosis,caveolin-dependent endocytosis,caveolae-dependent endocytosis and endosomes-dependent endocytosis respectively,and then the non-specific transmembrane mechanism of the disulfide bonds of FITC-PI were investigated.3.In the previous experiment,we found that the disulfide bonds of FITC-PI had specific transmembrane ablity to MDA-MB-231 and Calu-1 cells,and we hypothesized that there might have the same or similar membrane proteins that aid in the penetrating of MDA-MB-231 and Calu-1 cells.Cell membrane proteins of MDA-MB-231 and Calu-1 cells were extracted by Membrane Protein Extraction Kit and further analyzed by iTRAQ,membrane proteins of Human breast cancer MCF-7 cells were studied as control.And then the data were explored by bioinformatics databases and tools,and secretory carrier membrane protein 4(SCAMP4)was found to be the candidate for helping the disulfide bonds of FITC-PI in the.process of transmembrane access.Then,The COMPARTMENTS database was used to analyze the localization of SCAMP4 protein in the cells.SCAMP4 protein in MDA-MB-231 and Calu-1 cells were detected by RT-PCR and Western-blot.4.After the expression of SCAMP4 protein confirmed,total cytoplasmic protein data of MDA-MB-231 and Calu-1 cells were analyzed by Label free method,protein interaction network of SCAMP4 and the disulfide bonds of FITC-PI were further explored.Results:1.After 20 STRs and amelogenin gene loci of MDA-MB-231 and Calu-1 cells were detected,and the results indicated that MDA-MB-231 and Calu-1 cells keeping in our laboratory are the very cells without cross-contamination,and then we synthesizes the disulfide bonds of FITC-PI.And experiment established that the transmembrane access ability of the disulfide bonds of FITC-PI.2.In the study of nonspecific transmembrane transport mechanisms,we found that the disulfide bonds of FITC-PI can penetrate into MDA-MB-231 and Calu-1 cells by direct transport.The disulfide bonds of FITC-PI can be transfected into MDA-MB-231 cells through macropinocytosis,caveolin-dependent endocytosis,and endosomes-dependent endocytosis,the disulfide bonds of FITC-PI can be transfected into Calu-1 cells through macropinocytosis,and caveolae-dependent endocytosis.3.The membrane proteins of MDA-MB-231,Calu-1 and MCF-7 cells were extracted and analyzed by iTRAQ technique.Compared with MCF-7 cells,the specific membrane transporters detected in MDA-MB-231 and Calu-1 cells included B3KVN0,Q9ULF5,Q14332,B4DTS6,095864,P48509,SCAMP4,F8WDZ3,Q14114,B3KQX7,H0Y544,B7Z1M0,A0A0S2Z5F9 and C9K0C8.By Using the Uniport protein database,further results showed that secretory carrier membrane protein 4(SCAMP4)is the candidate for helping the disulfide bonds of FITC-PI in the process of transmembrane access.The COMPARTMENTS results showed that SCAMP4 protein can be expressed on the cytoplasmic membrane and little SCAMP4 can be detected on the Golgi apparatus.4.We further analyzed and identified all the cytoplasmic protein data of MDA-MB-231 and Calu-1 cells by Label free method.Then,string software were applied to construct protein interaction network of SCAMP4,by referring the cytoplasmic protein data of MDA-MB-231 and Calu-1,polyubiquitin C protein was found to be the downstream molecular of SCAMP4.And the further results suggested that the first transmembrane region of SCAMP4 is the key functional domain for the delivery of the disulfide bonds of FITC-PI in the process of transmembrane access.Conclusions:1.The disulfide bonds of FITC-PI can be transfected into MDA-MB-231 cells through macropinocytosis,caveolin-dependent endocytosis,and endosomes-dependent endocytosis,the disulfide bonds of FITC-PI can be transfected into Calu-1 cells through macropinocytosis,and caveolae-dependent endocytosis.2.Secretory carrier membrane protein 4(SCAMP4)is the candidate for helping the disulfide bonds of FITC-PI in the process of transmembrane access,and the expression of SCAMP4 are higher in MDA-MB-231 and Calu-1 cells compared with the MCF-7 cells.3.SCAMP4 is a specific membrane protein that facilitates the transduction of FITC-PI into the MDA-MB-231 and Calu-1 membrane.SCAMP4 locates on the cytoplasmic membrane and the first transmembrane region of SCAMP4 is the key functional domain for the delivery of the disulfide bonds of FITC-PI in the process of transmembrane access.4.Polyubiquitin C protein is the downstream molecular of SCAMP4.After the disulfide bonds of FITC-PI penetrating into MDA-MB-231 and Calu-1 cells,with the help of polyubiquitin C,the disulfide bonds of FITC-PI can delivery into the cytoplasm.