The Study Of Mitochondrial Genome And Proteomics On Platinum Resistance Cell Lines Of Ovarian Cancer And The Relationship To The Clinical Chemoresistance

Background and objective:The mortality of ovarian cancer is the highest among the gynecological malignant.The prognosis of ovarian carcinoma has been improved by chemotherapy based on cytoreductive(CRS),however,5 years survival rate of the advanced disease is still between 15-20%,the drug resistence of tumor cells is the one of the main causes.The known mechanisms of chemo-resistance of ovarian cancer included:1) Outflow of chemotherapy drug decreases concentration in cells.MDR1,MRP和LRP are the mostly involved resistant gene and protein;2) Conversion and detoxification of anti-tumor drug by carcinoma cells is enhanced,involving GSTs and P450;3) Chemotherapy drugs result in modification of target molecule on cells,including TOPⅡand reductase of dihydrofolic acid;4) Enhancement of DNA repair,including MGMT;5) Chemotherapy drug was unable to induce apoptosis of cancer cells.The present studies have revealed that expression of drug resistance genes of OC fails to predict the drug resistance and prognosis,the study of retroconversion of drug resistance fail to be an ideal approach, which indicates that there remains much unknown the mechanism of drug resistance.Mitochondria,which is a vital cell organella universally in eukaryocyte,,provides sites for oxidative phosphorylation and generation of ATP,and is responsible for 90%of the energy for celluler metabolism.Differing from other organella,it is distinctive for containing DNA and ability to transcription and translation.mtDNA can inherit,the transcription and translation of which is under the regulation of nuclear gene.Recent researches have indicated that mtDNA plays an important role in tumorigenesis,signal transmission and apoptosis.Mitochondria mainly composed of protien which accounts for 65%—70%dry weight of the total.As mtDNA contains limited information,the enzymes and structure protien underlying the 90%of cellular function depend on nuclear gene-coded.Oxidative phosphorylation and energy production in carcinoma cells are directly related to part of the mechanisms of drug resistance mentioned above,including(namely) drug decreased drug concentration in cells for outflow of chemotherapy and enhancement of conversion and detoxification of anti-tumor drugs.Moreover,Mt are involed in the apoptosis of tumor cells induced by p53.In addition,some of the drug resistance related enzyme are just located in mitochondria.Therefore,mitochondria deserve investigation concerning genome and proteome as it is associated with drug resistance.Funded by national natural science committee,our team has explored in the field of mitochondrial genome and its relation to drug resistance and prognosis of OC.10 specimens of mtDNA sequence of primary OC and recurrent(resistant) OC have been conducted and new polymorphism and 17 mutations(mutational rate 0.54/10,000bp) were identified.There is no significant difference between the mutational rate of mtDNA in initial treatment specimen and that in recurrent specimen.Considering individual variation among subjects and possible influence of primary resistance,it remains to be elucidated whether the mtDNA mutation is specifically related to drug resistance as well as the effects on expression of proteome are caused by these mutations.In the study,the cisplatin resistance cell lines and carboplatin resistance cell lines derived from platinum sensitivity SKOV3 and A2780 served as the models of drug resistance induced by chemotherapy in vitro.We studied the effects of chemotherapy resistance on mtDNA mutation and expression of proteome.We further verified different expression protein in drug-sensitive and resistant specimens under different clinical conditions.Material and Motheds1.Extract the mtDNA from SKOV3 line,A2780 line,cisplatin resistant cell lines and carboplatin resistant cell lines,and then amplified them by PCR for sequencing.Analyse the sequences of the chemotherapy sensitive cell lines and the chemotherapy resistant cell lines as well as their corresponding alterations of amino acids.Observe the effects of chemotherapy resistance on mtDNA mutation.2.Optimized isolation technology for mitochondria from cell lines:classical discontinuous density gradient centrifugation and repeatedly fractions centrifugation were introduced for isoltion of mitochondria from SKOV3,A2780,and their respeative cisplatin resistant lines and carboplatin resistant lines.Observation of the morphology and evaluation of purity was performed with electron microscopy and Western Blotting.3.DIGE was used for presenting protein expression spectrum of mitochondria from chemotherapy sensitive cell lines and chemotherapy resistant cell lines.Using sequence-specific proteases to break up the proteins into peptides on the gel was performed and A peptide-mass map was generated to detect different expression proteins by MALDI—TOF—MS (mass spectrometer).Search for possible candidate chemo-resistance associated protein in SwissPort and NCBInr databases.Verified the three differential expression proteins with Western Blotting.4.The expression of mitochondrial protein ATP-α,which had been down-regulated in the drug-resisted cell lines A2780/CDDP and A2780/CBP,was reevaluated in 21 chemotherapy-sensitive OC specimens and 21 drug-resistant OC specimens with immunohistochemistry.Results:1.Compared with the normal cells,31,34 and 38 mutations occurred respectively in the mtDNA of SKOV3,SKOV3/DDP and SKOV3/CBP lines,with5,8 and 10 corresponding amino acids changes,respectively.43,37 and 40 mutations occurred respectively in the mtDNA of A2780,A2780/ DDP and A2780/ CBP lines,with 9 corresponding amino acids changes respectively均为9。For the 6 cell lines,the mutation rate of the mtDNA was 22.4/10,000bp. After induction of drug resistance in vitro,SKOV3/CDDP presented 55 mutations in mtDNA compared with their mother generations(SKOV3);SKOV3/CBP presented 52(compared with SKOV3);A2780/CDDP presented 17(compared with A2780);and A2780/CBP presented 12 mutations(compared with A2780).2.Successful isolation of mitochondria from OC cell lines was obtained by both classical uncontinuous density gradient centrifugation and repeatedly fractions centrifugation. Western Blotting revealed satisfactory purity was revealed by western blot.Under the electron microscopy,high purity with more mitochondrial debris was observed with density gradient centrifugation while high purity with less mitochondrial debris with differential centrifugation.As more protein was extracted by fractions centrifugation,finally,this technology was adopted in our study.3.Combination of DIGE and MALDI—TOF—MS was introduced for compare-proteome analysis of the following lines:SKOV3 and SKOV3/DDP,SKOV3 and SKOV3/ CBP,A2780 and A2780/ DDP,A2780 and A2780/ CBP,and 236 differential expression spots were discovered including 108 up-regulation and 128 down-regulation. Nineteen differential expression spots,whose expression differences significant or presented in more than 2 lines,were selected for identification.13 protein spots were identified and 10 were down regulated,5 of which were mitochondrial protein,namely,ATP-α,PRDX3,PHB,ETF,ALDH,coded by nuclear DNA and responsible for oxidative metabolism and electron transport respiratory chain.Of the 5 proteins,ATP-αis the only one down regulated in two lines,A2780/CDDP and A2780/CBP,down regulation of PRDX3 and PHB in SKOV3/CBP has been verified by Western Blotting as well.4.ATP-αin ovarian cancer tissue was verified by Immunohistochemistry.No statistical difference(p=0.25) was found between the chemotherapy sensitive group and chemotherapy resistant group,despite that a higher ATP-αexpression was observed in the chemotherapy sensitive group.Conclusions:1.There were 31～41 mutations in mtDNA from sensitive ovarian cancer cell lines and resistant lines,more than those from ovarian cancer tissue.There were much more mutations of mtDNA of drug-resistant SKOV3 than drug-resisted A2780.2.High purity mitochondria were obtained from ovarian cancer cell lines by classical uncontinuous density gradient centrifugation and repeatedly frantions centrifugation. repeatedly fractions centrifugation is superior due to higher extraction with less mitochondrial debris.3.DIGE,characterized by integrity of 2-DE technique and CyDye DIGE fluors, successfully processed the comparative proteomic analysis with lower amount of mitochondrial protein.With a specific internal standard integration,the software automatically directs the rectification of expression of protein.Thus the results are more accurate,reliable and reproducible.We identified some differentl expression proteins,such as ATP-α,PRDX3,PHB,ETF,ALDH,which are coded by nuclear genes and all down regulated.These proteins are associated with energy metabolism and electron transfer respiratory chain.No proteins encoded by mtDNA was observed in the different expression proteins and it may be related to limited genetic informations in mtDNA and limitation by experimental techniques.4.Immunohistochemistry to detect ATP-αexpression in ovarian cancer tissue demonstrated a down -regulation of ATP-αin ovarian cancer tissue of patients with drug-resistance,however,without statistical significant differences.