Proteomic Analysis Of The Resistance In Human Lung Adenocarcinoma Cells To Cisplatin

BackgroundLung cancer is one of the common malignant tumor in the world. Reports from World Health Organization(WHO)suggest that lung cancer would be acting as the most killer for our health in the 21th century. Chemotherapy is the leading way to lung cancer with systemic treatment. Great improvements have made to the efficacy of lung cancer treatment with the combination of different anticancer drugs.Unfortunately, contemporary chemotherapies for lung cancer,usually containing cisplatin,demonstrate response rates in the 30-40%range.The overall 5 year survival rate of patients is less than 15%.Resistance to anticancer drugs is a major obstacle preventing the effective treatment of tumors.The Resistance to anticancer drugs mechanisms in lung cancer cells have been broadly explored,but they are still unclear.The phenomenon of the resistance is also known to be a multifactorial event in which several mechanisms act simultaneously.Recent years several mechanisms have been found to contribute to it,involving proteins such as P-gp(P-glycoprotein),MRP(multidrug resistance related protein), LRP(lung resistance related protein),BCRP(breast cancer multidrug resistance related protein),TOPâ…¡(topoisomeraseâ…¡)and GST (glutathione-S-transferase),etc.The main mechanisms include:tumor cells could greatly decrease the intracellular concentration of cytotoxic drugs,anticancer drugs could not arrive at their targets because of transportation of the intracellular cytotoxic drugs to other subcellular structures,structural alterations in the drug target enzymes and proteins increased their detoxification,and alterations in cellular metabolism enhanced the ability of tumor cells to repair DNA damage and resistant to apoptosis,etc.Although these pathogenesis studies on resistance to anticancer drugs in tumors have been undertaken successfully,the mechanisms are intricate and have not been fully elucidated yet. Proteomics,a new science which study total proteins of different stage in a cell,an organ or an individal with proteomic technique,provide a high throughout method to study the complexity of life.Through proteomics analysis,new targets or mechanism of drug-resistance may be found. Here we use proteomic tools to analysis the mechanism of resistance to anticancer drugs in human lung adenocarcinoma cell lines A549 and A549/CDDP in vitro.Partâ… Proteomic analysis of human lung adenocarcinoma cell lines A549 and A549/CDDP in vitroObjective To establish the two-dimensional gel electrophoresis (2-DE)patterns of human lung adenocarcinoma cell lines A549 and A549/CDDP and analysis the differentially expressed proteins between the two groups.Methods 1.A549 and A549/CDDP cell lines were cultured routinely with RPMI-1640 medium.MTT assay was performed to evaluate the resistance of A549/CDDP cell.2.Comparative two-dimensional gel electrophoresis(2-DE)technology was performed to separate the total protein of A549 and A549/CDDP,followed with modified colloidal Coomassie brilliant blue staining and Image Scanner scanning.3.PDQuest software was used to analyze 2-DE images. Result 1.The resistant index of A549/CDDP cell was 7.8.2.The well-resolved,reproducible 2-DE patterns of A549 and A549/CDDP were established.Fifteen differentially expressed protein spots were found between the two groups,of which 8 spots increased and 7 decreased in the expression levels in A549/CDDP group than in A549 group.Conclusion 1.Compared with A549 cell line,A549/CDDP has good resistance.2.The well-resolved,reproducible 2-DE patterns of A549 and A549/CDDP were established.Partâ…¡Identification and verification of differentially expressed proteins associated with A549 and A549/CDDP cell linesObjective To identify and verificate the differentially expressed proteins in cisplain resistance between A549 and A549/CDDP cell lines. Methods Differential expression proteins between A549 and A549/CDDP cell lines were identified by peptide mass fingerprint (PMF)based on MALDI-TOF-MS(Matrix-assisted laser desorption/ ionization time of flight mass spectrometry).These proteins were identified through searching SWISS-PROT and NCBInr dababase with Mascot software.ICC(Immunocytochemistry)and RT-PCR(Reverse transcriptase PCR)were used to verificate the differentially expressed level of several proteins between the two groups.Result 13 proteins were identified.Most of the identified proteins are correlated with tumur's biological prosperities of proliferation,invasion,apoptosis and immunity.The differentially expressed proteins could be divided into five main groups based on their functions:metabolic enzymes,proteins related to cellular structure,chaperones,proteins involved in drug detoxification or repair of DNA damage and proteins relative to signal transduction.Differentially expressed level of PKM₂(Pyruvate kinase M2)and CK18(Cytoskeletal 18)were further verified by ICC and RT-PCR.Conclusion 1.13 different expressive proteins between A549 and A549/CDDP cell lines,which may be related with cisplain resistance, were identified by peptide mass fingerprint(PMF)and bioinformatics analysis.2.The expression of these 13 proteins were verificated by ICC and RT-PCR,and the results were consistent with the ones of proteomic analysis. Partâ…¢Experiment Study on the relationship between cisplain resistance and suppression of PKM2 in A549 cell lines by small interfering RNAsObjective To analysis the relationship between cisplain resistance and suppressing PKM2 of A549 cell lines by vector based RNA interference.Methods A549 cell lines were cultured routinely. SiRNA-PKM2 was designed(SiRNA-PKM21 and SiRNA-PKM22)and was transfected into A549 cells medicated by cationic lipsome.There were 5 experimental groups:control,blank vector,SiRNA-neg, SiRNA-PKM21,and SiRNA-PKM22.1.The expression changes of PKM2 were assayed by immunocytochemical staining and RT-PCR.2. The cisplain resistance changes of A549 cells were evaluated by MTT. Result 1.The immunocytochemical staining and RT-PCR analysis revealed:â‘ suppression of PKM2 by SiRNA-PKM21 or SiRNA-PKM22 led to significant decrease in both protein and mRNA level of PKM2(vs.control,P＜0.05);â‘¡there were significant differences between SiRNA-PKM21 and SiRNA-PKM22(P＜0.05);â‘¢No significant difference was found among control,blank vector and SiRNA-neg(P＞0.05).2.suppression of PKM2 by SiRNA-PKM21 or SiRNA-PKM22 IC50 in A549 is 84.67μmol/L or 55.13μmol/L.The resistant significant increase.Conclusion 1.Suppression of PKM2 in A549 cell lines can be actualized by vector based SiRNA-PKM2.2. There are correlation between cisplain resistance and suppressing PKM2 of A549 cell lines by SiRNA-PKM2.