Screening And Identification Of Serum Protein Associated With Chemotherapy Resistance/sensitivity In Non-mall Cell Lung Cancer

Background:lung cancer is one of the most common malignant tumour in the world, accounts for 16% of all malignant tumour, 28% of all cancer deaths.Its morbidity has had the tendency to rise obviously The morbidity is rising year by year in China.According to statistics, the Mortality rate is 30.83/100,000 in China from 2004 to 2005.Non-small cell lung cancer is the main pathological types of lung cancer, accounting for 80%, while adenocarcinoma accounts for more than 35%~40%.Though The third Generation chemotherapy drug and target drug Emerge rapidly in the past 20 years, The total survival rate of lung cancer has not been obviously improved , about 14%.most probably, because it cann' t been diagnosed at early stage and Chemoresistance.70% of the NSCLC is already in advanced when found, which loses the opportunity to surgery.At present, the standard first-line treatment of advanced lung cancer-two platinum-drug program was only about 40% effective.In this study, 40 patients were enrolled retrospectively, the effective rate of 35% confirmed this.So finding the biomarkers to predict sensitivity/resistance in non-small cell lung cancer, is very important personalized medicine in clinical.People have been looking for an ideal method to predict the sensitivity of cancer chemotherapy.Common are:1.In vivo sensitivity test;2.In vitro sensitivity test;3.Detection of tumor markers;4.Detection of resistance genes.But chemosensitivity prediction methods have many shortcomings, is still not satisfactory, mainly in poor correlation with clinical and time-consuming issues such as long.Ideal method should:1.Good correlation with the clinical , sensitivity and specificity; 2.simple, rapid, time-consuming short; 3.Specimens easy to obtain, can be diverse; 4.Can be multi-drug screening; 5.Operable to standardization ; 6.low-cost,Drug resistance mutations in both genes, expression changes, or changes in drug pump or anti~apoptotic pathway appears to adjust resistance, who are the ultimate implementation of the protein.To find biomarkers at the protein level is great of significance, which can predict tumor resistance to chemotherapy .proteome means all proteins expressed by a piece of genome or a kind of cell / organization.Proteomics understands the interaction between proteins by analyzing the dynamic changes of the protein composition, expression and modification from an overall perspective.It can be said that the study of proteome is not only the milestone in life science research, but also one of the core content about life science research in post-genome era.Surface-enhanced laser desorption ionization time of flight mass stectrometry combines the energy absorbing molecule protein after excitation in laser ionization.The proteins of interest are captured on the chromatographic surface by adsorption, partition, electrostatic interaction or affinity chromatography depending on their properties, and analyzed by TOF mass spectrometry.The result is a mass spectrum comprised of the mass to charge {ml z) values and intensities of the bound proteins/peptides.Then, using these chromatographic surfaces TOF mass spectrometry.The result is a mass spectrum comprised of the mass to charge {ml z) values and intensities of the bound proteins/peptides.Then, using these chromatographic surfaces, a laser desorption time-of-flight mass spectrometer can generate an accurate protein profile of a biological sample requiring minimal amounts of sample.The protein sample may be analyzed directly or after proteolytic digestion of the adsorbed material.SELDI-TOF-MS technology is already widely used in a variety of diseases, espcielly in cancer.However, due to the limitations of Technology the sequence of protein peaks is unkwon.It is difficult for the the study of downstream.Clinport magnetic beads can purify the low abundance small molecular weight protein in serum.MALDI-TOF-MS was developed in recent years as a new type of soft ionization of biological mass spectrometry, The samples are mixed with an organic compound that acts as a matrix to facilitate thedesorption and ionization of compounds in the sample.The analyte molecules are distributed throughout the matrix so that they are completely isolated from each other.Some of the energy incident on the sample plate is absorbed by the matrix, causing rapid vibrational excitation.The analyte molecules can become ionized by simple protonation by the photoexcited matrix, leading to the formation of the typically singly charged ions.Some multiply charged ions are also formed, but this is rarer.The analyte ions are then accelerated by an electrostatic field to a common kinetic energy.If all the ions have the same kinetic energy, the ions with low mass tocharge ratio (m/z) travel faster than those with higher m/z values, therefore, they are separated in the flight tube and the number of ions reaching the detector at the end of the flight tube is recorded as the intensity of the ions.Objective:To Scan, purify and Identify the serum protein in non-small cell lung cancer patients, which can predict the chemosensitivity, by using SELDI-TOF-MS,Clinport magnetic beads and MALDI-TOF/TOF-MS.Material and Method:1.Patients choose 40 patients: From 2009 February to 2009 September, the lung cancer patients who see the doctor in the Affiliated Tumor Hospital of Guangzhou Medical College.According to the RECIST, 40 cases of patients with standard chemotherapy with platinum, divided into the chemotherapy valid set (CR +PR) and the Chemotherapy invalid set (SD+ PD).2.Processing and conservancy of the serum sample Extract 3~5 ml empty stomach vein blood of a sufferer in the morning with the dry tube, 4 "C, 1500 revolutions per minute for 10 minutes, withdraw serum, place them in -80 "C refrigerator.3.The SELDI-TOF-MS examination Combine reaction happens for a period of time after adding the sample to the top of the chip, various protein adsorb on protein chip by a particular group in the surface, then wash the non-special molecule that not combined with buffer liquid or water.Then add the energy absorption molecule aqua, being aqua aridity; we can put the chip to the chip reader to carry on a quality table analysis.Use the standard protein chips of the All-in-one as to outer quod vide.4.The statistics analysis4.1 Comparison of clinical data, using SPSS13.0 to analysis the difference between chemotherapy-sensitive group and resistance groups.Count data were analyzed by chi-square test, measurement data of non-normal distribution were analyzed by nonparametric rank sum test, normal distribution of measurement data were analyzed by t test, P <0.05 was statistically significant.4.2 using Protein Chip Software 3.land Biomarker Wizard software, According to analysis of variance principles calculation of chemotherapy-sensitive group and the resistance group the same mass to charge ratio between the peak of protein expression difference of P value.4.3 According to categorize decision tree principle, analyze the numeral group and the relativity with the Biomarker Patterns Software (BPS)and Establish the diagnosis medol.5. CLINPORT MB-WCX-2 bead separation and purification of small molecular weight protein6. MALDI-TOF/TOF-MS testThe sample was dissolved by 30% CAN completely, which contains 0. 1% TFA. And then , we used the Applied Biosystems 4700 plus MALDI-TOF-TOF Analyzer to analysis. The data of primary and secondary mass spectrometry was analyzed by GPS Explore software. We Search the local database to identify proteinsResults:1. There is no significant difference between sensitive group and the resistance group of non-small cell lung cancer in clinical characteristics.2. 100 valid protein peaks were found by SELDI-TOF-MS detection.3. 23 different protein peaks were found P <0. 05 between sensitive group of 14 case and resistance group of 26 cases in Non-small cell lung cancer who were treated with Standard two-drug chemotherapy with platinum.4. 19 different protein peaks were found P <0. 05 between sensitive group of 7 case and resistance group of 18 cases in Non-small cell lung cancer who were treated with Platinum and Taxol.5. 19 different protein peaks were found P <0. 05 between sensitive group of 7 case and resistance group of 18 cases in Adenocarcinoma of lung cancer who were treated with cisplatin and docetaxel. 6. 9 protein peaks were High expression in resistance group, mass charge ratio were 3193. 14Da, 3263. 61 Da, 3397. 71 Da, 5902. 89 Da, 6087.73 Da , 9993.63 Da, 2950.49 Da, 6108.91 Da and 4993.65 Da.7. By uniting protein peaks of 3193. 14Da and 3263.61 Da , the prediction model was established to predict the chemosensitivity of non-small cell lung cancer, who were treated with the joint difference in protein can the standard chemotherapy with Standard two-drug chemotherapy with platinum, the sensitivity was 90.23% and specificity was 98.56%.8. Clinport magnetic beads was an effective and feasible dection for purifying low abundance of small molecular weight protein in serum9. The protein peaks of the 3193. 14Da, 3263. 61Da and 3397. 71Da were identified by MALDI-TOF/TOF-MS. The protein sequences were TCSIEAHVAGLPPIMSNSAETTASAVTGAARandNAANKPMPTDALPVAAPSAPHIDIGIKDADRK Matching proteins were not found in the database.Conclusions:1. SELDI-TOF-MS is the Effective tool to find the biomarkers to predict the chemosensitivity of non-small cell lung cancer.2. The prediction model can be established to predict the chemosensitivity of non-small cell lung cancer by SELDI-TOF-MS and Bioinformatics software3. Expression of serum protein was different between sensitive group and resistance in non-small cell lung cancer who were treated with Standard two-drug chemotherapy with platinum.4. 9 protein peaks were High expression in resistance group, mass charge ratio were 3193. 14Da, 3263. 61 Da, 3397. 71 Da, 5902. 89 Da, 6087.73 Da , 9993.63 Da, 2950.49 Da, 6108.91 Da and 4993.65 Da.5. By uniting protein peaks of 3193. 14Da and 3263.61 Da , the prediction model was established to predict the chemosensitivity of non-small cell lung cancer, who were treated with the joint difference in protein can the standard chemotherapy with Standard two-drug chemotherapy with platinum, the sensitivity was 90.23% and specificity was 98.56%.6. Clinport magnetic beads was an effective and feasible dection for purifying low abundance of small molecular weight protein in serum7. The protein peaks of the 3193. 14Da, 3263. 61Da and 3397. 71Da were identified by MALDI-TOF/TOF-MS. The protein sequences were TCSIEAHVAGLPPIAASNSAETTASAVTGAARandNAANKPAAPTDALPVAAPSAPHIDIGIKDADRK Matching proteins were not found in the database.