| Background and AimsEsophageal carcinoma is one of the most common cancers in the world. Anestimated482,300new esophageal cancer cases and406,800deaths occurred in2008worldwide. Incidence rates vary internationally by nearly16-fold, with the highest ratesfound in Southern and Eastern Africa and Eastern Asia and lowest rates observed inWestern and Middle Africa and Central America in both males and females. The incidencerates of esophageal carcinoma ranks the ninth in cancer, and mortality rate ranks the eighthin cancer in the world. Esophageal cancer is3to4times more common among males thanfemales. In Chinese mainland, an estimated259,000new esophageal cancer cases and211,100deaths occurred in2007. The incidence rates of esophageal carcinoma ranks thefifth and mortality rate ranks the fourth in cancer in Chinese mainland. Esophageal cancerusually occurs as either squamous cell carcinoma in the middle or upper one-third of theesophagus, or as adenocarcinoma in the lower one third or junction of the esophagus andstomach. In the highest risk area, stretching from northern Iran through the central Asianrepublics to North-Central China, often referred to as the ''esophageal cancer belt,''90%ofcases are esophagus squamous cell carcinomas(ESCC).Despite advances in surgical therapy for ESCC, the overall prognosis of patients hasnot significantly improved over the last few decades due to locally-advanced ordisseminated diseases most patients present at diagnosis, half of which die in recurrence ormetastasis even after combined therapy including surgery, chemotherapy and radiationtherapy. For the patients diagnosed at early stage, the five-year survival rate after therapyachieves90℅～100℅, however, the five-year survival rate after therapy lower than10℅for the patients diagnosed at advanced stage. The survival benefit for the patientsdiagnosed at early stage is because treatments are more effective at this early stage. It istherefore vital to diagnose oesophageal cancer at an early stage, before the development ofsymptoms, when treatment can dramatically improve prognosis.Cytological screening techniques have been developed for the early detection ofESCC and have been used particularly in the Linxian region of China, where there arelarge numbers of people who are asymptomatic, but at high risk of ESCC. This cytologicaldiagnosis has low sensitivity(14℅～36℅)for the detection of cancers, which is taking place in preference by biopsy and endoscopic screening. An ideal technique to determinehigh risk of progression to cancer would not only be sensitive and specific, but would alsobe quick, non-invasive and would not require specialist interpretation. Endoscopic biopsysurveillance does not meet this ideal on many levels. It is invasive and expensive, biopsytaking is laborious and samples only a small area of the heterogeneous epithelium andthere is inter-observer variation in the time-consuming pathological interpretation.However, multiple biopsies, which have been fixed in formalin, do allowimmunohistochemistry to be performed. Immunohistochemistry can inform about thepresence of certain biomarkers, their specific location within the tissue and provide somequantitative information,which shows to be sensitive and specific for diagnosis.Immunohistochemistry for MCM-2and cyclin-A have been used, for example, to predictrisk of progression to advanced cancer. However, the immunohistochemistry biomarkersfor diagnosis at early stage with high sensitivity show to be scarcity. It is urgent to identifynew immunohistochemistry biomarkers for early diagnosis of ESCC.Modern, high-throughput proteomic methods provide one way of profiling thesignificant changes in protein expression of tumor samples with respect to control, usingtissue biopsies obtained from patients diagnosed with this disease. Proteomic screeningtechniques are particularly useful for furthering the understanding of the mechanisms thatunderlie complex phenotypes like ESCC, in that they provide the information, which maycontribute to the diagnosis,molecular therapy and prognosis. Differential tagging withisotopic reagents, such as ICAT or the more recent variation that uses isobaric taggingreagents (iTRAQ), followed by multidimensional LC and MS/MS analysis is emerging asone of the more powerful methodologies in the searchfor disease biomarkers. Our recentstudies using both iTRAQ reagents resulted in identification and relative quantification ofproteins leading to discovery of potential cancer markers for colorectal cancer. Herein wehave extended the use of iTRAQ labeling in combination with multidimensionalLC-MS/MS analysis to ESCC for comparison of protein profiles of ESCC andnon-cancerous oesophagus tissues in an attempt to identify potential biomarkers, as well asto identify in a global fashion molecular pathways that are deregulated in ESCC, which inturn should aid in drug target discovery. MethodsThe cancer tissue samples and the paired normal tissue samples of five cases of earlystage ESCCs were collected. After total protein extraction, the five cancerous smples andthe paired normal samples were pooled and trypsin digested, respectively. Then biologicalsamples were labeled with isobaric mass tags for relative and absolutequantitation(iTRAQ), and pooled. Multidimensional liquid chromatography, and massspectrometry has been used to analyze the labeled biological samples to identify proteinsthat are differentially expressed in human eraly stage ESCC and paired normal mucosa forcancerous biomarker discovery. Cancer and non-cancerous samples were compared byon-line and off-line separation. Protein identification was performed by mass spectrometry.The difference protein spectrum was constructed. The location and function of differentialproteins were analyzed by searching Swiss-Prot and GeneOntcology(GO) database.Based on bioinformatics analysis, a group of different proteins were selected forfurther validation by imunohistochemistry. The cancer tissue samples and the pairednormal tissue samples of57cases of early stage ESCCs were collected. Theimunohistochemistry were performed through the following streamline operation: fixed byformalin→embeded by paraffin wax→sliced→deparaffinage to water→antigen restore→endogenous peroxydase sealed by hydrogen peroxide→incubation with primary antibody→incubation with secondary antibody→coloration with DAB→counterstain withhematoxylin→dehydration→transparention. Two pathological doctors were invited todiagnose the results double blindly. The mmunohistochemical localization was scored in asemiquantitative fashion incorporating both the intensity and the distribution of specificstaining. The extent percentage of positive cells was graded as follows:0, negative;1,<10%positive cells;2,11%to50%positive cells;3,51%to80%positive cells;4,>80%positive cells. The staining intensity was graded as follows:0, negative;1, weakly positive;2, moderately positive; and3, strongly positive.The cancer tissue samples and the paired normal tissue samples of3cases of earlystage ESCCs were collected. After total protein extraction, Western-Blot analysis wasperformed to for further validation. Thirty microgram of total proteins were used. AfterSDS-PAGE→trarsmembrane→incubation with primary antibody→incubation withsecondary antibody→electrochemiluminescence→development→fixation→scanning→semi-determinalion analysis by PD QUEST software, the different expression ofpotential biomarkers were validated. ResultsA newly developed iTRAQ technique was used in the present study to compareprotein expressions between ESCC and matched normal mucosal tissue. A total of712proteins were identified in both runs. Using cutoff values of1.5-fold for up-regulation and0.67-fold for down-regulation,29overregulated and10down-regulated proteins wereidentified in ESCC samples compared with normal mucosal tissues. According toannotations from the UniProt knowledgebase (Swiss-Prot/TrEMBL) and the GeneOntology (GO) Database,71.79%of the39differential proteins were located in more thanone cellular component. In brief,25.46%,30.77%,66.67%, and41.3%of the proteins werelocated in the extracellular region, plasma membrane, cytoplasm, and nucleus of cells,respectively. The following biological processes were observed to change more frequently:protein binding (including protein or peptide binding, RNA or mRNA binding, andcadherin binding)(74.35%), enzymes (30.77%), regulation (30.77%), cell structure(23.08%), and signal transduction (20.51%)(Figure3). In total,79.48%of the39differential proteins were involved in more than one biological process and molecularfunction.Among the differentially expressed proteins, TP-alpha, Collagen alpha-1(VI) chainand S100A9were verified to be upregulated in77.19%,75.44%and59.65%of ESCC byimmunohistochemistry and western-blot. Cutoff values for TP-alpha (HADHA), Collagenalpha-1(VI)(Col6a1) and protein S100A9were determined as scores2,3and3,respectively, to determine ESCC from normal tissue by comparing the sums of sensitivityand specificity of various diagnosis criteria. TP-alpha shows a sensitivity of77.19℅and aspectivity of100.00℅, Collagen alpha-1(VI) chain(COL6A1) shows a sensitivity of71.93℅and a spectivity of85.96℅. Protein S100A9shows a sensitivity of82.46℅and aspectivity of43.86℅. Furthermore, upregulation of TP-alpha (HADHA), Collagenalpha-1(VI)(Col6a1) and protein S100A9were validated by Western-Blot analysis.Diagnostic Value of TP-alpha, Collagen alpha-1(VI) and protein S100A9werevalidated. These results provide new insights into ESCC biology and potential diagnosticand therapeutic biomarkers, which suggest that TP-alpha, Collagen alpha-1(VI) chain andS100A9are potential biomarkers of ESCC, and may play an important role intumorigenesis and deveopment of ESCC. ConclusionsAn iTRAQ-based difference expression proteomic research on ESCC was performed.A total of39disregulated proteins were identified, and a new set of up-regulated proteins,HADHA, Col6a1, and S100A9, were validated by Western blot and immunohistochemistry.These results provide new insights into ESCC biology and potential diagnostic andtherapeutic biomarkers, which suggest that TP-alpha, Collagen alpha-1(VI) chain andS100A9are potential biomarkers of ESCC, and may play an important role intumorigenesis and deveopment of ESCC. |
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