Preliminary Proteome Study And Clinical Diagnosis Investigation Of Giant Cell Tumor Of Bone

Introduction Giant cell tumor of the bone (GCTB) is primary boneneoplasm, there are many multinucleated giant cells which are considered to beformed from mononuclear stromal type tumor cells, are not true tumor cells forthe reason that they do not have proliferative activity, while fibroblast-likemononuclear stromal cells are real tumor cells. GCTB is often considered aproblematic neoplasm because it has the potential for malignancy, a high rate oflocal recurrence, and the ability to metastasize. Cell and molecular biology studyindicate: The common abnormity of GCTB chromosome are aneuploidy, haploid,translocation of chromosome, tip deletion, end to end fusion, invertedduplication, telomeric association, unidentified additional material on the longarm. Some authors believe relapse and translocation of GCTB may be due totelomerase activity and telomeric weaken. The areas of mononuclear stromalcell and it's nuclear, mitosis are higher in the relapse group than that innon-relapse group. Tumor genesis of GCTB is correlated to oncogenes. Tumortissue at the site of cortex or cancellus bone connection, especially in cortex isdifficult to curetted, even with the help of additional or adjuvant methods oftreatment in order to facilitate treatment and prevent any possible recurrence. Itis also difficult to kill all the tumor cells. Leap tumor focus may be formed at thesoft tissue or cancellus bone connecting site, which causes the post-operationrelapse.Tumor proliferation, relapse and translocation are closely correlated withblood supply, invading of tumor cells into blood and vessel wall is one of thereasons of translocation. There are plenty of capillary vessels in the peripheralpart, which is obviously more than center part of GCTB, and grow flourishingly.While there are fewer capillary vessels which are thick or end-closed, and evenno vessels in center part. Vascular endothelial growth factor (VEGF), a potentangiogenic factor, promoting penetrating of vessels, as a potent stimulator oftumor angiogenesis, the level of it's expression may provide some prognosticindication of biologically aggressive behavior and local disease recurrence inosteolytic lesion of GCTB, matrix metallo-proteinase (MMP) implicated in theprogression of solid tumors is indispensable for local recurrence. VEGF andMMP-9 expression correlate with the extent of bone destruction and localrecurrence, MMP over-expressed and tissue inhibitors of metallo-proteinases(TIMP) low-expressed may regulate pathological osteolysis of bone, and may beresponsible for local recurrence and translocation, and that the expression ofthese cytokines may correlate with the extent of bone destruction and localrecurrence. MMP-9 thaws the matrix membrane outside of cells, promotingvascular endothelial cells migrating and angiogenesis. Over-expression ofMMP-9, MMP-2 and low-expression of TIMP-1, TIMP-2 cause pathologicalosteolysis, and correlate with the local recurrence of GCTB. The number ofMMP-9 positive cells are higher in the recurrence or translocation group thanthat of the non-recurrence group, it may therefore provide some prognosticindication.In previous study, scholars have know some proteins which haveaggressive characters, then study the expression of them in GCTB usingRT-PCR, immuno-histo-chemistry and in situ hybridization assays. It is almostimpossible to know well the whole information. Proteome, a new technologymake it is feasible to analyses qualitatively and quantitatively of proteins in cellscomprehensively. The term 'proteome' was defined as the entire proteincomplement expressed by a genome or a cell or tissue. The proteome is dynamicbecause the protein complement changes with time and according tophysiological needs of the organism. Proteomics approaches will certainlyadvance our understanding of basic mechanisms that are altered in the complexprocesses leading to carcinogenesis,progress and transforming. Diagnosticproteins may be found through comparing the proteins expressed by normal andpathological cells quantitatively and qualitatively, which may indicatepredisposition to specific carcinogen effects, forecast potential markers,administer in effective therapies and enable more rational designs for clinicaltrials.Objective To search and study proteins responsible for aggressivecharacter of GCTB with proteomic techniques, to identify the dependabilitybetween image and pathology, the accurate of ultrasonic puncture biopsy, toidentify the effectiveness of image bonding ultrasonic puncture biopsy in thediagnosis of bone tumor.Methods 5 cases of aggressive GCTB(Jaffe gradeⅡ-Ⅲ,Campanaccigrade Ⅲ,G0-1T1-2M0) and 4 cases of benign GCTB (Jaffe grade Ⅰ,Campanacci gradeⅠ, G0T0M0)were selected as experimental and control grouprespectively to find the differential protein expression by isoelectric focusing /SDS acrylamide gel two-dimensional electrophoresis, then the differentiallyexpressed proteins were identified by mass spectrographic analysis, in this experimentwe selected 3 proteins stains to authenticate.We collected the plain films, CT and MRI of 23 patients who werediagnosed giant cell tumor of bone with plain film, then analysised withpathology to analysis the signs of plain film, CT and MRI of giant cell tumor ofbone, simple bone cyst, aneurysmal bone cysts, bone fibrous dysplasia, tosummarize the outlines of differentiation of them.We compared the result of ultrasonic puncture biopsy diagnosis with theresult of image diagnosis and post-operation pathology diagnosis of seventeenpatients.Results 506±23 protein stains were found in experimental group, 468±28protein stains were found in control group by comassie brilliant blue staining,there was significant difference between the two groups on two-dimensionalelectrophoresis(P<0.05).Up-regulation of Annexin A1 and 14-3-3 proteinε,down-regulation of Peroxiredoxin 1 were found in experimental group.Fifteen cases were diagnosed of giant cell tumor of bone by pathology inthe whole twenty-three cases(Jaffe gradeⅠfour cases, Jaffe grade Ⅱnine cases,Jaffe grade Ⅲtwo case), the total nicety rate of giant cell tumor of bone were65.2% (15/23) by x-ray. Two cases were at proximal part of humerus, threecases were at distal part of humerus, five cases were at distal part of femur, threecases were at upper end of tibia, one case was at lumber vertebral body3, onecase was at ilium. Five cases were diagnosed bone cyst, one case was at upperend of humerus, one case was at olecranon, one case was at the distal part ofradium, one case was at distal part of femur, one case was at ilium. One casewas diagnosed aneurysmal bone cyst at proximal humerus. Two cases werediagnosed fibrous dysplasia, one at distal part of radia, one at calcaneum. Sevencases were diagnosed of giant cell tumor of bone by x-ray, four wereauthenticated of giant cell tumor of bone, two were authenticated of bone cyst,one was authenticated of fibrous dysplasia, the nicety rate was 57.1%. Ninecases were more inspected by CT after examination of x-ray, which werediagnosed of giant cell tumor of bone, but only seven cases were authenticated,the other two were bone cyst, the nicety rate were 77.8%. Five cases were moreinspected by MRI after examination of x-ray, three cases of giant cell tumor ofbone, one case of bone cyst, one case of aneurysmal bone cyst, were diagnosedrespectively, which were identical to pathology, the nicety rate was 100%. Onecase of giant cell tumor of bone and one case of fibrous dysplasia werediagnosed by CT and MRI after x-ray examination, which were identical topathology.Eight cases were diagnosed of giant cell tumor of bone by puncture biopsy,but twelve cases were diagnosed by image, the according rate was 66.7%,whilenine cases were authenticated by pathology, the according rate was 88.9%.Three cases of bone cyst, Two cases of fibrous dysplasia, one case of lungcancer metastasis, two cases of osteosarcoma were authenticated by bothpathology and puncture biopsy. One case was diagnosed aneurysmal bone cystby image, but no blood-like fluid was harvested, one case was diagnosed cancermetastasis both by image and puncture biopsy. One case was diagnosed fibrousdysplasia by image, but two cases were diagnosed by puncture biopsy, theaccording rate was 50%. One case was diagnosed osteosarcoma by image, buttwo cases were diagnosed by puncture biopsy, the according rate was 50%. Onecase was diagnosed bone cyst by image and puncture biopsy, pathology.Discussion Annexins are a family of proteins that bind to phospholipidsand carbohydrates in a calcium-dependent manner. Annexin A1, an anti-inflammatory protein, is now recognized as an important signal in a variety ofother systems. Lipopolysaccharide (LPS) markedly activated the Annexin A1gene in epithelial cells, neutrophils, and peritoneal, mesenteric, and alveolarmacrophages cell types known to function in experimental endotoxemia.Administration of LPS to AnxA1-deficient mice produced a toxic responsecharacterized by organ injury and lethality. Annexin A1, an anti-inflammatoryprotein was undetectable in lungs and pancreas of cftr(-/-) mice, tissues knownto express cystic fibrosis transmembrane conductance regulator (CFTR),absence of this inhibitory mediator of the host inflammatory response mayaggravate cystic fibrosis. Annexin A1 play a role in apoptosis caused byCaspase. Down-regulation of Annexin A1 in head and neck cancer is associatedwith epithelial differentiation status. Annexin A1 is highly expressed inWell-differentiated head and neck squamous cell carcinomas (HNSCCs),whereas very weak or negative expressed in moderately and poorlydifferentiated HNSCCs. Distinctive loss of Annexin A1 gene and Annexin A1protein is found in breast cancer, suggests it's involvement in maintainingnormal breast biology. Annexin A1 up-regulated was found in aggressive GCTBwhich has more mononuclear stromal cells, these results suggest that annexinA1 may be a key protein involved in cystic fibrosis pathogenesis,anti-inflammation and in improving organism recovery. We suggest thatincreased expression of annexin A1 may contribute to assisting apoptosis andpreventing even more worsening of aggressive GCTB.Peroxiredoxin I, a member of the peroxiredoxin family, a scavenger ofreactive oxygen species, reduces peroxides and equivalents through thethioredoxin system. Increased DNA damage and tumor susceptibility inPeroxiredoxin 1 (prdx1)-/-mouse results from peroxiredoxin 1 down-regulation.peroxiredoxin 1 is up-regulated in esophageal squamous cell carcinoma.Expression of Peroxiredoxin I was associated with local recurrence and lymphnode recurrence, Peroxiredoxin I expression indicates tumors with a highpotential for recurrence in tongue cancer. P53 up-regulated modulator ofapoptosis (PUMA) induced apoptosis is dose and time dependent, and is directlyassociated with reactive oxygen species (ROS) generation;overexpression ofPUMA induced up-regulation of peroxiredoxin 1. Peroxiredoxin 1 expressionwas down-regulated in interdigital cells at embryonic day 13.5, in which dayinterdigital cells committed to programmed cell death (PCD) in mouse, the levelof NF-kappaB inhibitor epsilon is dramatically reduced, while an increase inNF-kappaB activation and reactive oxygen species levels can be found in thecytoplasm in Peroxiredoxin 1 gene knock-down cells. There was down-regulation of Peroxiredoxin 1 in aggressive GCTB, then may be much moreROS was produced in it, DNA is much more prone to be damnified, unbalanceof oncogene and anti-oncogene may cause the aggression of GCTB.14-3-3 Proteins, a family of ubiquitous phosphoserine/threonine-scaffoldproteins which have seven isoforms (beta, gamma, epsilon, eta, sigma, theta andzeta) play important roles in a wide range of vital regulatory processes,including signal transduction, apoptosis, cell cycle progression and DNAreplication.14-3-3 epsilon, one isoform of that family organizes cell signalingand is involved in exocytosis. Coexpression of 14-3-3 epsilon with eukaryotictranslation initiation factor 2 alphas (eIF2) alpha leads to the abrogation of theinhibitory effect of eIF2 alpha on TGF beta-mediated signaling. The 14-3-3epsilon protein is one of the caspase-3 substrates, cleavage of 14-3-3 proteinduring apoptosis promotes cell death by releasing the associated Bad from the14-3-3 protein and facilitates Bad translocation to the mitochondria and itsinteraction with Bcl-x(L). 14-3-3 epsilon is highly expressed in both lung cancercell lines and normal lung fibroblasts, it may be involved in lung cancertumorigenesis. 14-3-3 epsilon was highly expressed in aggressive GCTB, signaltransduction and apoptosis may be improved. Putrescence and cystic fibrosis inaggressive may be due not only to faster growth without enough blood supply,but also to apoptosis mediated by 14-3-3 proteinε.Of course, proteome is a developing subject which needs to be perfected intechnique and theory, image diagnosis theory and experience are still needed tobe consummated summarized, additional cases with longer period ofobservation are needed to be studied. However, This paper may alert theclinician who may consider additional or adjuvant methods of treatment in orderto facilitate treatment and prevent any possible local recurrence or metastasis todiagnosis more nicely.Conclusion Non-linear IPG strips and optimized SDS acrylamide gel mayimprove the separation of whole proteins in cells, sample preparation andelectrophoresis condition were the key events for two-dimensional gelelectrophoresis.The down-regulation of Peroxiredoxin 1 may be responsible for theaggression of GCTB, the up-regulation of Annexin A1 and 14-3-3 proteinεmay be due to the result of reaction of organism. This change may cause GCTBto be more aggressive, it may be useful to diagnosis, these three proteins may be·16·selected as tumor markers of the aggressive GCTB.Colligation of plain film, CT and MRI, combining with clinical behaviorsand general status of patients may advance the diagnosis and discrimination ofgiant cell tumor of bone. Plain film is easily managed and cheaper, may providethe whole signs, it can satisfy the need of orientation and partly determination ofnature, while CT and MRI can provide more information of the lesion. In somedegree, image may provide determinate reference, ultrasonic puncture biopsyhas a high right diagnosis rate. Image combined with ultrasonic puncture biopsyis a good choice for the diagnosis of bone.