Comparative Mitochondrial Proteomic Analysis Of Raji Cells Exposed To Adriamycin Or Radiation And Relevance With Chemoresistance

BackgroundMuch progress in conventional therapy for NHL have been achieved over the past decades,but there are still 40%to 70%of patients with intermediate and high grade NHL failing to achieve long-term disease-free survival(DFS),and no curative treatment strategies have been established for patients with low grade NHL yet.In this study,the reason for selecting Raji cells is that the Raji model mirrors human lymphomas that have mutant p53 and increased BCL2 expression,which are commonly present in patients with NHL and are considered a source of chemotherapy failure in these patients associated with chemoresistance.Chemotherapy is commonly used as part of the treatment against non-Hodgkin's lymphoma(NHL) and can be advantageous for some time.Adriamycin(ADR) is widely used in drug combination strategy like CHOP(Cyclophosphamide+Adriamycin+Vincristine+Prednisolone) against NHL as a classic anthracycline agent,while some patients have shown resistance to it.The chemoresistance and anticipated dose-related cardiotoxicity associated with ADR are critical obstacles to a successful outcome. Multidrug-resistance(MDR) is a common pattem of anti-chemotherapy in tumor cells. The known mechanisms of MDR included:1) Outflow of chemotherapeutic drug decrease concentration in cells.MDR1,MRP and LRP are the mostly involved resistant genes and proteins;2) Conversion and detoxification of anti-tumor drug by carcinoma cells is enhanced,including GSTs and P450;3) Chemotherapeutic drugs result in modification of target molecule on cells,including TOPâ…¡and reductase of dihydrofolic acid;4) Enhancement of DNA repair,including MGMT;5) Chemotherapeutic drug was unable to induce apoptosis of cancer cells.Lymphoma is a category of radiosensitive malignance and radiation therapy(RT) alone may be used as a curative treatment for stageâ… andâ…¡in patients with indolent non-Hodgldn's lymphoma(NHL).For more extensive and aggressive NHLs, RT is used in combination with chemotherapeutic agents,while 50-70%of these patients relapse.The aim of radiotherapy on NHL is to induce apoptosis with the endpoint of proliferative cell death(PCD,also known as "secondary apoptosis") and lead to unrepaired DNA injury.NHL is more radiosensitive than most solid malignancies while resistance to radiotherapy is a major obstacle to a successful outcome.The cellular radiation response is complex and apoptosis is the main purpose for radiotherapy to suppress the proliferation of NHL cells,so mitochondrial proteins play crucial role in the radiation signal transduction pathway due to their vital function in apoptosis.Mitochondria are eukaryotic organdies that play a crucial role in several cellular processes,including energy production,fatty acid metabolism,oxidative stress,ion homeostasis and programmed cell death.The main chemical components of mitochondrion are protein and lipide,accounting for 65-70%and 25-30%of the total dry weight of mitochondia respectively.Mitochondria is wrapped by an outer and inner membrane,having four domains including outer-membrane,inner-membrane, intermembrane space and ground substance.In mitochondria of a liver cell,the content of protein of the above-mentioned four parts are 8%,21%,4%and 67% respectively.The inner membrane is especially rich in proteins,e.g.the high molecular weight multi-protein-complexes of the respiratory chain are located at the inner mitochondrial membrane.The total number of different proteins or polypeptides making up a mitochondrion is estimated to be around 2000～2500.Mammalian mtDNA encodes for 13 essential polypeptide components of the oxidative phosphorylation system,small and large rRNA's,and 22 tRNA's.The remaining mitochondrial proteins of the oxidative phosphorylation system,metabolic enzymes, membrane channel proteins,and any other protein regulating mitochondrial function are derived from the nuclear genome.Import of these proteins into the mitochondria is highly regulated through the function of protein chaperones and the inner and outer tr nsmembrane peptide import complexes(TIM and TOM).In recent years,with the development of proteomics technology,it becomes more and more achievable to have a thorough investagtion of comparative proteomics.For instance,many new significant proteins have been discovered with diverse physiological and pathophysiological effects.Two-dimensional gel electrophoresis (2-DE) is currently the mainstay of proteomic analysis as it allows the simultaneous visualization of thousands of proteins and their post translational modifications (PTM's)(12).It separates a complex protein mixture based upon 2 intrinsic protein characteristics,net charge(isoelectric point,pI) and molecular mass.First,proteins are separated according to their net charge though isoelectrie focusing(IEF) and they are subsequently separated orthogonally by molecular mass using sodium dodecylsulfate-polyacrylamide gel electrophoresis(SDS-PAGE).Although theoretically 2-DE has no difficulty in separating all the proteins,it still has many disadvantages,such as time-consuming,sensitivity,linear dynamic range, repeatability,quantitation truth(precision/accuracy) and compatibility with mass spectra.In addition,2-DE is not good at detecting proteins with low abundance,high molecular weight(＞200KDa) or the minimum molecular weight(＜10KDa) and extreme acid and basylous proteins.Two-dimensional fluorescence different gel electrophoresis(2D-DIGE) is the new development of 2-DE,it also allows for the direct comparation of protein abundance changes which is less than 10%across multiple samples simultaneously with 95%statistics reliability coefficient without interference due to gel-to-gel variation.Now many new data were added into forthcoming databases such as SWISS-PROT,NCBI and MitoPeoteome,ect. MitoP2 is the most comprehensive database about mitochondrial proteome which includes the.explanation of moiety on human,mouse and yeast.The proteins above-mentioned of the three parts are 624,615,522 respectively.However,due to the spurt of the emergement of this new subject,the databases are still very insufficient and require more exploration to enrich them.Partâ… Comparative Mitoehondrial Proteomie Analysis of Raji Cells Exposed to AdriamycinObjectiveIn this study,we used Raji cells as a model of human NHL.We aimed to investigate ADR about its antineoplastic activity,drug resistance and unexpected toxicity on non-Hodgkin's lymphoma(NHL) Raji cells at mitochondrial proteomic level.Methods1.Selection of the dose and time exposure of ADR.NHL Raji cells(4×10⁵) were exposed to ADR(0.2,0.5,1.0,1.5,2.0μg/ml respectively) for the various incubation times indicated(24 h,48 h,and 72 h).The result of the cell proliferation evaluated by the MTT was exhibited as the inhibition rate.The IC50 values were determined directly from the semi-logarithmic dose-response curves.2.Mitochondria isolation and purification validation.ADR(1.5μg/ml) was added to the 2×10⁷ Raji cells at the initiation of the 48 hour culture period.The selection of the dose and time of exposure was based on the MTT results.At this stage, the mitochondria of Raji cells were isolated using a mitochondrial isolation kit according to the manufacturer's instructions.Separated mitochondria were mounted on glass slides,incubated with 0.1%Janus green B solution for 10 min,and then visualized under a light microscope.Purified mitochondrial pellets were preserved at -80℃until further analysis.The purity of the isolated mitochondria was validated by western blot.COXIV,PCNA,and Cathepsin D were used as mitochondria,nuclear and lysosomal markers,respectively.3.Two-dimensional differential in-gel electrophoresis and imagine.Protein concentrations determined by Bio-Rad protein assay for the control and ADR-treated groups were used to normalize the quantities of protein loaded in each sample. Aliquots of 100μg protein from each of the two samples and labeled with 200 pmol of either Cy3(treated) or Cy5(control) respectively.The Biological Variation Analysis(BVA) module of Decyder 6.5 was used to compare the control and test sample to generate list of differentially expressed proteins.Taking cut-off of 1.5-fold up/down-regulation with a t-test score p＜0.05 as an initial threshold for significance.4.LTQ-ESI-MS/MS analysis and database searching.Total 26 protein spots were cut out of 2D-DIGE gels using a Gelpix Spot-Excision Robot and the digested pieces were analyzed via LTQ-ESI-MS/MS using a surveyor high-performance liquid chromatography(HPLC) system.All identified proteins were determined to be mitochondrial-associated proteins by inquesting several protein databases such as Swiss-Prot,NCBI,EMBL,Biolnformatic Harvester,WOLF PROST,Target P, MitoSub,and MitoP2.Results1.The result of MTT indicated that_incubation of Raji cells with ADR showed that inhibition rate was in a dose-and time-dependent manner.It reached 49.82%with 1.5μg/ml ADR treatment for 48 h.Based on these result,we used 1.5μg/ml of ADR for 48 h for further experiments. 2.Mitochondria Isolation and Purity Validation.To obtain mitochondria of Raji cells with high purity for the reliabilities of proteomic analysis,Janus green B staining was carried out to identify the mitochondrial fraction and western blot analysis was applied to validate its purity.The isolated mitochondria were stained as bluish-green round particles.COXIV was specially detected in the purified mitochondrial fraction and this fraction lacked any detectable contamination of abundant nuclear and lysosome proteins such as PCNA and Cathepsin D.Our result demonstrated a high purity of mitochondria with the use of our subcellular isolation method.3.Isolation of mitochondrial proteins exposed to ADR.Total 1485 spots were detected,and 63 were differentially expressed(≥1.5-fold).4.Mass spectrometry identification of differentially expressed mitochondrial proteins.We chose 26 spots for further analysis by mass spectrometry and identified 37 unique proteins.Among them,34 proteins decreased and 3 proteins increased.The altered proteins identified in this study encompass a range of mitochondrial functions including OXPHOS,cell cycle regulation,transporters and channels,DNA repair, redox,protein synthesis and degradation,ect.A relative large percentage of the proteins have slightly basic pI value range of 5-9(32 out of 37,86.49%).About 75.68%(28 out of 37) proteins identified have masses between 10-60kDa.Partâ…¡Bioinformatics Analysis of Proteins Associated with Chemoresistance of NHL and Apoptotic Pathway InterferenceObjectiveIn this part,we aimed to establish biomolecular interactive networks including several identified mitochondrial proteins with multi-informatics techniques.By using the specific apoptotic pathway inhibitor,the mitochondrial proteins associated with the chemoresistance will be explored as potential anti-tumor targets in NHL treatment.Methods1.Validation of selected identified proteins by western blot.To further validate the alterations in protein abundance derived from 2D-DIGE/MS,we examined the variance of three enzymes of interest(HSP70,PHB,and ABCB6) by western blot. These proteins were chosen due to their crucial functions in the action of ADR on Raji cells and the commercial availability of the corresponding antibodies. 2.To study functional interactions and possible pathways of the HSP70,PHB,and ABCB6,Pathway Studio 5.0 software(Ariadne Genomics,Rockville,MD,USA) was used.Briefly,we firstly import a protein list including HSP70,PHB,and ABCB6 into a new pathway diagram,and build a pathway using the option "Find all entities connected to selected entities(Expand Pathway)".The program will search the current pathway database and ResNet for interactions with the selected entities and add them to the pathway.3.LY294002 was added to Raji cells prior to ADR.The inhibited effect of LY294002 was evaluated by analyzing several proteins and genes closely associated with chemoresistance such as HSPA9,PHB,P53 and AKT.The proliferation of Raji cells treated with LY294002 was evaluated by MTT analysis.Cell cycle of the treated Raji cells and the control were determined by flow cytometry using PI staining.Results1.Validation of HSP70,PHB,and ABCB6.Comparing mitochondrial lysates from control and ADR-treated Raji cells by Western blot,the expressions of HSP70 and PHB were increased whereas the expression of ABCB6 was decreased.The results were in conformity with 2D-DIGE.2.The software Pathway Studio 5.0 was used to search possible protein-protein interactions,common regulators,cell processes,and related diseases for HSPA9,PHB, and ABCB6.By this approach,we found that proteins belonged to different structural and functional families had PHB as common target and were involved in mitogenesis, defense response,germination,inflammation,proliferation,and apoptosis,ect. Furthermore,most of the diseases associated with these three proteins were cancer derived from various organs.3.The pathway studio software construct interactive network within the AKT pathway.Then two proteins associated with chemoresistance including HSPA9 and PHB were investigated in detail at mRNA level and protein level using real-time PCR and western blot analysis.Both HSPA9 and PHB were up-regulated in ADR-treated Raji cells.After treated with LY294002,an inhibitor for PI3K,the expressions of these two proteins were down-regulated with a significant alteration in cell cycle and up-regulation of p53. ObjectiveThe aim of this present investigation is to get a comprehensive overview of the protein turnover reactions in mitochondria of Raji and discover molecular biomarkers in radiation response.Methods1.Irradiation procedures:Exponentially-growing Raji ceils were suspended at a concentration of 1×106 cells/ml in 1640 and irradiated at room temperature using a X-ray source(16 MV,CLINAC2100C/D Linear Accelerator,Varian,USA) at a dose rate of 200 cGy/min.Single doses of 2,4,and 6 Gy were given and the cells were subsequently cultured at various times(24,48,and 72 h) for analysis.2.MTT assay:Cell proliferation was evaluated by MTT.3.Apopotosis assessment of 2-Gy X-ray on Raji ceils by Annexin V/PI double staining:To assess apoptosis,Raji ceils exposed to 2 Gy X-ray for 48 h and its control partner were stained with FITC-conjugated Annexin-V protein along with PI according to the recommendations of the manufacturer and analyzed by flow cytometry.4.Comparative mitochondfial proteomic analysis of Raji cells exposed to radiation: 2D-DIGE in combination with LTQ-ESI-MS/MS were utilized as a non-biased approach to evaluate mitochondfial protein alterations of NHL Raji cells after exposed to radiation.5.Validation of Selected identified Proteins by Western Blot:To further validate the alterations in protein abundance derived from 2D-DIGE/MS,we examined the variance of two interested enzymes(RECQL4,and ATAD3B) by Western blot.These proteins were chosen clue to their crucial function in the action of radiation on Raji ceils and the commercial availability of correspondent antibodies.The expression levels of all tested proteins were in conformity with 2D-DIGE(Figure 4).Results1.The result of MTT indicated that_incubation of Raji ceils with various dose radiation showed that inhibition rate was in a dose-and time-dependent manner.2.Apoptosis analysis of Raji cells exposed to various dose radiation:the apoptotic response to radiation treatment is lower if compared to the control Raji cells.3.Isolation and identification of mitochondrial proteins exposed to radiation.Total 1391 spots were detected,and 34 were differentially expressed(≥1.5-fold).We chose 26 spots for further analysis by mass spectrometry and identified 23 unique proteins. Among them,19 proteins decreased and 4 proteins increased.The altered proteins identified in this study encompass a range of mitochondrial functions including OXPHOS,energy metabolism,cell cycle regulation,DNA repair,protein synthesis and degradation,ect.4.Validation of RECQL4,and ATAD3B:Comparing mitochondrial lysates from control and radiation-treated Raji cells by Western blot,the expressions of RECQL4 increased whereas the expression of ATAD3B was decreased.The results were in conformity with 2D-DIGE.Conclusions1.In this study,2D-DIGE combined with mass spectrometry and database interrogation were used to simultaneously screen the mitochondrial protein abundance changes of Raji cells exposed to ADR and radiation.The results of our proteomic analysis indicated that our study strategy was suitable and high performance.2.A series proteins associated with chemoresistance relevant to ADR including HSP70,PHB,and ABCB6 were found to be altered in their expressions.However, such markers require functional studies before being introduced to the clinic.4.In the PI3K/AKT pathway,HSPA9 and PHB were closely associated with chemoresistance.The expression of these two proteins could be influenced by LY29.5.Proliferation of Raji cells,shown in our model by the MTT,Annexin V/PI analysis and proteomic study,was not significantly slowed down and the apoptotie response to radiation treatment is lower if compared to the control Raji cells.6.GAPDH,RECQL4,MKI67,and ATAD3B could be used as promising biomarkers for radioresistance.