ITRAQ Based Screening Of Multi-drug Resistance Related Proteins In Gastric Cancer

Gastric cancer is the second leading cause of cancer related death in theworld, and the third in China, posing a great threat to human health.Chemotherapy is one of the major treatments of gastric cancer. Multidrugresistance, or MDR, is the major cause of chemotherapy failure. We previouslyidentified a group of multidrug resistance related proteins using the two cellmodels of7901/ADR and7901/VCR. However, the two drugs are seldomlyused in clinic, and the mechanisms of MDR remains to be elucidated. Highthroughout techniques can be used to screen multi-drug resistance relatedproteins and explore the mechanisms of gastric cancer drug resistance.Proteomics based iTRAQ is an excellent choice for studying the MDRmechanisms.We set out to apply iTRAQ into MDR research to screen for multi-drugresistance related proteins in three newly established drug resistant gastriccancer cell lines, in the hope of finding some drug resistant targets.AimTo establish three drug resistant gastric cancer cell sublines using SGC7901 as parent cells. Use proteomics-based techniques to screen for key drugresistance related proteins.MethodsSuspension cultured SGC7901cells established by our lab were regularlycultured, and exposed to escalating dose of5-fluoracil, cisplatin, epirubicinrespectively to establish gastric cancer drug resistant cell sublines. The startingconcentration of5-fluoracil, cisplatin, epirubicin are0.4μg/ml,0.08μg/ml,4ng/ml, respevtively. MTT, apoptosis assay, adriamycin accumulation andretention assay, immunofluorescence, western blot, migration and invasion assaywere used to study the biological functions of the newly established drugresistant sublines. Screening of drug resistance related proteins was carried outusing iTRAQ. The identified proteins were further analyzed by bioinformaticsoftware MetaCore.Results1. Three gastric cancer drug resistant cell sublines,7901/5FU,7901/CDDP,7901/EPI, were successfully established. The three sublines not exhibited greatdrug resistant capabilities，with Resistance Index（RI）being5.2，9.6and11.6,respectively, but also showed different drug resistant properties to differentchemotherapy.2. The drug efflux functions of7901/5FU,7901/CDDP,7901/EPIwere0.40±0.02（p<0.05），0.19±0.03（p>0.05）,0.36±0.02（p<0.05）, comparedwith parent releasing index of0.14±0.01. ABC binding cassette proteins, such asMRP1was found to be associated with it. Moreover, the three drug resistant cellsublines exhibited increased resistance to apoptosis. Western blot demonstratedthat upregulation of Bcl-2(p<0.05) and downregulation of Bax (p<0.05) mayexplain some of the mechanisms.3. F-actin based morphological change of7901/5FU,7901/CDDP,7901/EPIcells was observed. Epithelial markers such as E-cadherin, β-catenin was highly expressed in parent SGC7901, while mescenchymal markers vimentin, slug,snail was remarkably increased in7901/5FU,7901/CDDP,7901/EPI cells,demonstrating obvious EMT phenotype in the three drug resistant cell lines. Andthis kind of drug induced EMT promoted migration and invasion of the cells.4. Two biological replicates of iTRAQ analyses were carried out.5930proteins were found in the first iTRAQ analysis. Compared with parentSGC7901, the common proteins that increased more than1.2or decrease below0.8in7901/5FU,7901/CDDP,7901/EPI cells, with p value less than0.05, wereregarded as significant proteins.18upregulated proteins and33downregulatedproteins were identified for the first iTRAQ.5586proteins were found in thesecond iTRAQ analysis.19upregulated proteins and25downregulated proteinswere identified for the second iTRAQ. Upregulation of9proteins anddownregulation of10proteins were found when we chose intersection of thetwo results. Proteins of intersection were analyzed by informatics. Unfoldedproteins response and cytoskeleton change were identified to be some of themechanisms of gastric cancer drug resistance. And endoplasmin, VCP mightinvolve in the drug resistance regulating proteins as a molecular chaperone.Vimentin and keratin may affect the cytoskeleton of the cell to decrease drugsensitivity.Conclusion1. Three drug resistant cell sublines,7901/5FU,7901/CDDP,7901/EPI, weresuccessfully established.2. Increased drug efflux function and apoptosis resistance may partly explaintheir drug resistant properties.3. Epithelial to mescenchymal transition was observed. And increasedmigration and invasion capabilities, indicating that chemotherapy may inducedEMT to facilitate tumor progression, apart from killing most tumor cells.4. iTRAQ based screening is useful in discovering gastric cancer drug resistance related proteins. Endoplasmin and VCP were found to be the keydrug resistance related molecules in gastric cancer. Vimentin may affect thecytoskeleton of cancer cells to modulate drug sensitivity. Expression of vimentinwas confirmed by western blot. However, further expressional and functionalvalidation is needed for further studies.