Proteomic Analysis Of Ubiquitination-Associated Proteins Inahuman Lungadenoc-Inomacisplatin-Resistant Cellstrain

ObjectivesThe objective of this study was to provide novel experimentalevidence at the proteomic level for the exploration of the changes in UPP incisplatin-resistant lung cancer cells by screening forubiquitination-associated proteins involved in cisplatin resistance in ahuman lung adenocarcinoma cell strain using a comparative proteomicstrategyMethods1. Estimating drug resistance model. Observating the drug resistance,morphological, Cell cycle and apoptosis by optical microscope, electronmicroscope, MTT assay, and flow cytometry,Which would Estimate IfA549/CDDP was a successful drug resistance.2. Employing1D SDS PAGE to separate ubiquitinated proteinsisolated and enriched from A549and A549/CDDP lysates via affinitychromatography. The differentially expressed bands between45-85kDawere subsequently hydrolyzed by trypsin and subjected to HPLC-CHIP-MS/MS analysis.3. Screening meaningful protein by Western Blot and confocal lasertechnology to verify the MS analysis of the results.Results1. After treating both cell strains with CDDP, the density of A549cellsdecreased remarkably, shrank and became round in shape and exhibitedtypical apoptotic morphological characteristics. The growth and apoptosisof A549cells underwent significant change. What,s more, the number ofA549in G2phase increased.However, No marked change occurred inA549/CDDP cells.2. The results of HPLC-CHIP-MS/MS show that In the11proteinsidentified,7proteins were monoubiquitinated or polyubiquitinatedsubstrates, and4proteins were E3ubiquitin ligase-associated proteins. The4proteins were RNF6, LRSAM1, TRIM25and PKM2.3. The results of Western blotting and confocal laser scanningmicroscopy indicated that the expression levels of the E3ubiquitin ligasesRNF6, LRSAM1, and TRIM25in the A549cell were obviously lower thanthose in the A549/CDDP cell line. The expression levels of the above3ubiquitin ligases in both cell lines were significantly down-regulated upontreatment with Cis-Diamminedichioroplatinum (CDDP), and the expressionin the A549/CDDP cell after the treatment with CDDP decreased to a lesserextent. The expression of the substrate PKM2in the A549cell was higher than that in the A549/CDDP cell. Moreover, the expression of PKM2wasup-regulated in the A549cell line and down-regulated in the A549/CDDPcell line upon CDDP treatment.ConclusionsThis study suggests that drug resistance is closely correlated withchanges in the ubiquitination process at the protein level in a human lungadenocarcinoma cell strain.