The Clinical Significance Of Candidate N-linked Glycoprotein Expression In Esophageal Squamous Cell Carcinoma

BackgroundEsophageal cancer(EC) is the eighth most common malignancy with a very poor prognosis. The mortality of EC in the world was 407 000 deaths in 2008, of which half occurred in China. Histologically, esophageal squamous cell carcinoma(ESCC) accounts for the majority of EC worldwide. The global incidence of ESCC shows considerable geographical variations. One of the well-known high risk areas EC is the so-called Asian esophageal cancer belt extending from eastern Turkey to Northern and Western areas of China. Other major high incidence areas for EC include Hong Kong, Japan, Southeastern Africa, Southern France and parts of South America. In contrast, esophageal cancer is the fourth most frequently diagnosed cancer and the fourth leading cause of cancer death in China, with an estimated 259 235 new cases and 211 084 deaths in 2008. EC is characterized by its high mortality and the five-year survival rate of ESCC is 15%-25%. The main reason for such poor prognosis is that EC cases at early stage are asymptomatic and go undetected until they have spread beyond the esophagus and are unresectable. In this setting, successful strategies of early detection, early diagnosis and early treatment for EC are critically needed to control this disease.Protein glycosylation accounting for 70% of human proteome is one of the most common post-translational modifications. Sugar chains of glycoprotein plays important physiological functions such as cellular recognition, intercellular and celluar-matrix interaction. Abnormal protein glycosylation is involved in the formation and progression of many diseases. Our previous study has identified 22 high-mannose proteins and 23 sialic proteins with differential expression by two-dimensional electrophoresis and mass-spectrometric technique. This present study further verified the differential expression and clinical significance of seven proteins in ESCC.AimsThe present study aims to verify the differential expression of seven candidate glycoproteins in ESCC tissue and serum samples, and to determine their clinical value, which would provide experimental evidence for identification of ESCC biomarkers with high sensitivity and specificity. In addition, the dysregulated glycoproteins may shed lights on the formation and development of ESCC underlying the molecular mechanisms of ESCC.Methods1 After selection of 80 pairs of ESCC and adjacent non-tumor tissue samples comprising 20 cases of each TNM stage with no statistical difference in terms fo age and sex, protein was extracted followed by protein concentration measurement by Bradford assay.2 Senenty-two serum samples including 72 ESCC, 48 esophagitis and 24 cases with upper gastrointestinal complaints but normal epithelial histology were selected from our serum bank for Western blot verification or followed by N-glycoprotein extraction using chromatography column with Concanavalin A(Con A), Lentil lectin(LCH) and Snowdrop lectin(GNA).3 Protein was separated by SDS-PAGE of 12% separation gel and 4% stacking gel and transferred to PVDF membrane. The membrane was blocked with 5% non-fat milk and incubated with first and secondary antibody followed by ECL detection of targets.4 Image Master software was used for semi-quantitative analysis of Western blot bands.5 Statistical software SPSS 17.0 was used for statistical analysis. Student’s t Test was used for statistical significance analysis with P < 0.05 regardsed as significance.Results1 HP expression decreased significantly in ESCC of TNMⅠ-Ⅲ stages(P < 0.05). The expreeion of HP in TNM Ⅰ stage was the lowest and increased gradually from TNM Ⅰto Ⅳ.2 In ESCC of TNMⅢand Ⅳstages, procathepsin D(p CD) expression was significantly higher than normal adjacent non-ESCC tissue, while in ESCC of TNMⅠand Ⅱstage, p CD also has the tendency of up-regulation but without statistical significance.3 In ESCC of TNM Ⅰstage, superoxide dismutase 3(SOD3) expression was significantly higher than adjacent non-ESCC tissue but only showed the tendency of enhancement in ESCC of TNM Ⅱ and Ⅲ stages.4 The expressions of complement C3(C3), PRELP and Clusterin(CLU) in ESCC showed no consistency in ESCC of each clinical stages.5 The serum level of HP expression increased significantly in ESCC serum compared with those of esophagitis and esophageal mucosa normal cases(P < 0.05). In contrast, the serum level of clusterin was significantly lower than those in esophagitis and esophageal mucosa normal cases(P < 0.05).6 N-glycosylated HP enriched by lectin chromatography from serum displayed remarkable increase compared with neat HP serum level, in particular in cases whose serum level of HP showed undectable differential expression.Conclusions1 Alterated expression of HP may be involved in the formation and progression of ESCC and serum HP or N-glycosylated HP may have the potential of becoming ESCC serum biomarker but further research is warranted for confirmation.2 PCD may play a role in the middle and advanced stage of the malignant progression of ESCC which involves infiltration, metastasis.