Mitochondrial Proteomics Of Ovarian Cancer, Paclitaxel-resistant Cell Lines

Background and objectiveOvarian cancer ranks the third in incidence of gynecologic malignancies and claims the highest mortality rate.Its five-year survival rate is only 15-20%.Chemotherapy resistance has been identified as one of the most important reasons for such low survival.Known mechanisms of chemotherapy resistance include:1) Chemotherapy drugs outflowing reduces the intracellular drugs concentration.Related genes and proteins include:MDR1,MRP and LRP et al.2) The antitoxic ability of carcinoma cells has improved.GSTs and P450 family are the related genes.3)The target that chemotherapy drugs work on has changed,such as topoisomeraseⅡ(TOPOⅡ),dihydrofolate reductase (DHFR).4) Repairing ability for DNA damage has been promoted.For example, MGMT activity has been enhanced.5) Chemotherapy failed to induce the apoptosis of cancer cells.P53 and Bcl-2 family are the related genes.Theoretically speaking,the expression of genes related to chemotherapy resistance in ovarian carcinoma cells could predict chemotherapy resistance or prognosis of the carcinoma.However,most reports failed to have the expected theoretic outcome.The reasons could be as follows:the tumor genesis needs the cooperation of both nucleus and cytoplasm,and expression of oncogenes of nuclear genome is regarded as the main reason. An interesting phenomenon has been found in several studies that emerging healthy cytoplasm and carcinoma nucleus could inhibit the development of carcinoma,indicating presence of anti-carcinoma products outside the nucleus.In eukaryotic cells,Mitochondria are critical subcellular organelles responsible for ATP generation through oxidative phosphorylation.It is the only place in addition to nuclei where DNA could be generated,transcripted and translated.Mitochondrial DNA(Mt DNA) can influence the anti-carcinoma ability of cells.At least three of the five chemoresistance mechanisms are related to mitochondria,such as drugs outflowing mechanism,improved antitoxic ability and apoptosis change of the carcinoma cells.Thus mitochondria have been selected as our study object.Proteomics approach provides a method for evaluating the expression of objective proteins,measuring the modified protein after translation and acquiring the chemoresistance related proteins concurrently,which offers a great opportunity to obtain a new marker of the malignancies and carry out early diagnosis of the disease.As the main component of mitochondria is protein,proteomics approach could comprehensively evaluate the association between mitochondria and ovarian chemoresistance and may acquire targets of the chemoresistance.Based on the identification of ovarian carcinoma chemoresistance genes,we have isolated and purified mitochondria from taxol sensitive and taxol resistant ovarian carcinoma cell lines by repeated fractions centrifugation and had them verified with electronic microscope and western blot method.Then,an Guanidination modified acetyl stable isotope labeling and LC-FTICR MS(liquid chromatography—hybrid linear ion trap fourier-transform ion cyclotron resonance mass spectrometry) strategy was performed to get the whole expression profiling of ovarian carcinoma mitochondria protein,as well as the different expression protein between chemotherapy sensitive and taxol resistant cells.Materials and methods:1.Measurement of chemoresistance of taxol resistant cell lines and evaluation of influence of taxol on MMP.Cultivated of the ovarian chemosensitive cell lines SKOV3,A2780 and corresponding chemoresistant cell lines SKOV3-TR,A2780TR and measured their resistance index with CCK-8 methods.After stimulating these cells with taxol of different concentration and staining them with JC-1 dyes,we examined the mitochondria membrane potential with fluoroscopy and Flow Cytometry to determine the influence of taxol on mitochondria function of different chemosensitive and chemoresistant cells.2.Optimized the methods of isolation of mitochondria from cell lines.Kit lysis method, classic fraction centrifugation combined with discontinuous density gradient centrifugation,and repeated fractions centrifugation were employed to isolate mitochondria from ovarian carcinoma taxol sensitive cell lines SKOV3,A2780 and their taxol resistant cell lines SKOV3-TR,A2780-TR,and mitochondria purity was verified by electron microscope and Western blot.3.Reportedly first use of Guanidination modified acetyl stable isotope labeling and LC-FTICR MS strategy to obtain the whole expression profiling of ovarian carcinoma mitochondria protein and the different expression protein between chemotherapy sensitive and taxol resistant cells.Result:1.The chemoresistant cells used in the experiment maintained excellent taxol resistant characteristics and the primary cell shape.The resistant indexes of SKOV3-TR and A2780-TR are 8.34±4.31 and 5.37±4.26,respectively.After being stimulated by 25uM taxol,the decreased mitochondrial membrane potential could be identified by fluorescent microscope and FCM.when the taxol concentration was 25uM,the taxol sensitive cell line SKOV3 exhibited significant change of MMP(P＜0.01).However, the taxol resistant cell line did not show a significant change at the same taxol concentration.It suggests that the mitochondrial functions of taxol sensitive cells and resistant cells influenced by taxol were different and mitochondria may be one of the important targets of taxol resistance.2.Comparing the methods of isolation of mitochondria,kit lysis method failed to show normal shape and only had cracked pieces with double membrane;the purity of mitochondria was about 40-50%.Much better isolation effects could be achieved by both optimized repeated fractions centrifugation and classic fractions centrifugation combined with discontinuous density gradient centrifugation.The purity of mitochondria could be up to about 70%.Next,efficiency of the two methods was evaluated.By repeated fractions centrifugation,1 mg mitochondria could be obtained from 2x10~8 SKOV3 and SKOV3-TR cells or 3-4x10~8 A2780 and A2780-TR cells.By fractions centrifugation combined with discontinuous density gradient centrifugation, only 200-300ug mitochondria could be isolated from the same amount of cells and the reproducibility of this method was worse.Therefore we selected repeated fractions centrifugation in our experiment and examined the isolated mitochondria with mitochondria marker COX4,nuclear marker Lamin-B,cell membrane marker Flotillin-1 and cytoskeleton proteinβ-actin by western blot method.It was shown that there were almost no contaminations of nuclei,cell membranes and cytoplasms.3.For the first time,Guanidination modified acetyl stable isotope labeling and LC-FTICR MS strategy was used to obtain the whole expression profiling of ovarian carcinoma mitochondria protein and the different expression protein between chemotherapy sensitive and taxol resistant cells.About 780 proteins have been identified in total and 240 proteins of them are shared by the two groups.Eight proteins were significantly altered in both kinds oftaxol resistant cell lines(＞2 fold or＜0.5 fold).In addition,28 proteins showed trends toward significant alterations(1.5-2 fold or 0.67-0.5 fold).Among them,nuclei encoded mitochondrial protein Mimitin (Myc induced mitochondria protein) is the product of oncogene Myc transcripted, which could be one of the proteins associated with taxol resistance.Conclusion:1.The chemoresistant cell lines used in the experiment maintained excellent taxol resistant characteristics and the primary cell shape.The MMP of taxol sensitive cells and resistant cells influenced by taxol was different and mitochondria may be one of the important targets of taxol resistance.2.Both classic fractions centrifugation combined with discontinuous density gradient centrifugation and optimized repeated fractions centrifugation could produce mitochondria of high purity,however,the latter method had better efficiency and reproducibility.After being identified by western blot,there were no contamination of nuclei,cell membrane and cytoplasm.3.Guanidination modified acetyl stable isotope labeling and LC-FTICR MS strategy was performed to obtain the whole expression profiling of ovarian carcinoma mitochondria protein and the different expression protein between chemotherapy sensitive and taxol resistant cells.Mimitin expression decreased in both SKOV3-TR and A2780-TR cells and it could be one of the proteins associated with taxol resistance.