Expression Of Annexin-1and VEGF-C In Esophageal Squamous Cell Carcinoma

Background and ObjectivesEsophageal cancer is a common malignant tumor of digest system.The number of the new cases is approximately480thousand,and the number of the death cases is400thousand per year all over the world. Its incidence is significantly different in varies areas, and developing countries have a higher incidence than that in developed countries.China is one of the highest incidence countries of esophageal cancer.The number of the new cases is above250thousand each year in china,and the areas mainly distribute in Henan、Shanxi and Hebei province around the Taihang mountain, the north of Jiangsu area,Chaoshan area and the north of Sichuan basin.The pathology of most esophageal cancer is Squamous cell carcinoma,which accounts for above95%. Epidemiological studies have shown that the occurrence of esophageal cancer closely associated with age, gender,race,genetic factors, eating habits, living environment, geographical environment, occupation, etc. With the development of molecular biology, studies have proved that the occurrence and development of esophageal cancer was regulated by a variety of oncogene and anti-oncogene which affect the expression of these genes, and many factors were interacting with each other, which resulted in Esophageal cancer.Annexin-1is one member of Annexins family,and it has the most characteristic of Annexins.It is also the best-studied protein in Annexin family. Annexin-1is known as lipocortin-1、ANXA-1、calpactin-1、endonexin or p35, and it is the first protein member that has been cloned in Annexin family.The encoding gene of Annexin-1is located in9p12～q21.2in human chromosome,including thirteen exons and twelve introns,containing1041bp in the open reading frame.Annexin-1has346amino acids,and its molecular weight is37kDa. Annexin-1is a mutiftmtion protein,and it participates in many important biological activities,such as anti-inflammatory action、 inducing cell differentiation、cell attaching and migratemg、cell proliferation and apoptosis、signal transmission,etc.Vascular endothelial growth factor C(VEGF-C) was first found in nutrition solution of prostate canner cell by V.Joukov in1996.It can stimulate VEGFR-3which is known as Flt-4,which can make tyrosine phosphorylate. Its gnen is located in4q34of human chromosome,containing2400bp.VEGF-C contains386amino acids,and its molecular weight is42.9kDa.As the first founded lymphangiogenic factor,it is considered as the single specific lymphangiogenic factor to act on lymphatic endothelial cell,and mostly relates on lymph node transfer of tumor cell.At present, domestic studies have not reported the correlation between the expression of Annexin-1and VEGF-C in esophageal carcinoma at the same time, and the relationship between the expression of them and the progression, invasion and metastasis of esophageal cancer is not very clear. This experience wants to detect the expression of Annexin-1and the VEGF-C protein in esophageal squamous cell carcinoma cells, and explores the mechanism in the formation of esophageal cancer as well as the relationship between the clinical and pathological features of esophageal cancer, analyses the correlation between the two protein for the propose to find a new method for guiding the early diagnosis and clinical treatment of esophageal cancer.Materials and Methods1. MaterialsThe resection specimens of esophageal squamous cell carcinoma (ESCC) and adjacent normal esophageal tissue were collected in the thoracic surgery of The Second Affiliated Hospital of Zhengzhou University from July2011to July2012, including80cases ESCC tissue and80cases adjacent normal esophageal tissue. All cases had not been preoperative radiotherapied and chemotherapied.2. MethodsEach specimen was fixed by formaldehyde solution, then embedded in paraffin. Each serial slice was4mm thick. Using SP immunohistochemical stained each slice.Immunohistochemistry steps were operated according with reagent box instructions.3. The results of immunohistochemistryThe expression of Annexin-1was mainly located in membrane and cytoplasm, and cell nucleus had a small amount of coloring. It was considered positive when cell membrane and cytoplasm were brown; VEGF-C protein expression was mainly located in the cytoplasm,and it was considered positive when cell cytoplasm contained brownish yellow granules substances. Each specimens was selected in10high-power field at random, counting100cells per field, according to the percentage of positive cells and the color depth. A:the percentage of positive cells<25%for0point,25%～for1point,51%～for2points,≥75%for3points;B:the staining intensity colourless for0point, light yellow for1point, yellow for2points,deep yellow for3points. Finally, A+B≥3points was considered positive expression.4. Statistical methodsThe data were analyzed by SPSS17.0software. Using χ2test of categorical data analyses the expression differences of Annexin-1and VEGF-C protein between ESCC and adjacent normal esophageal tissue and the relationship between the expression of Annexin-1and VEGF-C and the clinicopathological features in ESCC, applying the coefficient of contingency of correlation evaluates Annexin-1and VEGF-C protein expression in ESCC.All the inspection levels were a=0.05.Results1. The expression of Annexin-1in the adjacent normal esophageal tissue and ESCC, and the relationship with clinicopathological parameters.The positive rate of Annexin-1protein expression in ESCC was38.75%(31/80), lower than the adjacent normal esophageal tissue83.75%(67/80), the difference was statistically significant (χ2=34.128, P=0.000).The positive rate of Annexin-1in the ESCC group, age less than or equal to55-year-old group was35.71%(10/28), age above55-year-old group was41.18%(21/52),P=0.683; male group was40.00%(20/50), female group was36.67%(11/30), P=767; high differentiation group was45.45%(15/33), middle differentiation group was35.71%(10/28), low differentiation group was31.58%(6/19), P=0.564; the group that tumor did not beyond submucosa of esophagus was50.00%(4/8), the group that tumor invased muscularis layer of esophagus was45.00%(9/20), the group that tumor invased the outer membrane and beyond of esophagus outer membrane was34.62%(18/52)^P=0.568; lymph node metastasis group was42.86%(18/42),without lymph node metastasis group was34.21%(13/38), P=0.428.2. The expression of VEGF-C in the adjacent normal esophageal tissue and ESCC,and the relationship with clinicopathological parametersVEGF-C protein positive expression in ESCC was71.25%(57/80), higher than the adjacent normal esophageal tissue46.25%(37/80), the difference was statistically significant (χ2=10.316, P=0.001).The positive rate of VEGF-C in the ESCC group, age less than or equal to55-year-old group was60.71%(17/28), age above55-year-old group was76.92%(40/52), P=0.127; male group was78.00%(39/50), female group was60.00%(18/30), P=0.085; high differentiation group was72.73%(24/33), middle differentiation group was71.43%(20/28), low differentiation group was68.42%(13/19), P=0.947; the group that tumor does not beyond submucosa of esophagus was62.50%(5/8),the group that tumor invased muscularis layer of esophagus was70.00%(14/20),the group that tumor invased the outer membrane and beyond of esophagus outer membrane was73.08%(38/52), P=0.819; lymph node metastasis group was95.24%(40/42), without lymph node metastasis group was44.74%(17/38), P=0.000.3. The relationship between Annexin-1and VEGF-C expression in ESCC.In the ESCC group,the expression of both Annexin-1and VEGF-C were positive in9cases,both were negative only in1case, the expression of Annexin-1was negative and the expression of VEGF-C was positive at the same time in48cases, the expression of Annexin-1was positive and the expression of VEGF-C was negative at the same time in22cases.The correlation of the expression between Annexin-1and VEGF-C were positively correlated (r=-0.596, P=0.000).Conclusions1. The expression of Annexin-1and VEGFC was correlated with the occurrence and development of esophageal cancer.2. The expression of Annexin-1showed no significant association with age, gender, the degree of differentiation, the deepen of tumor invasion, lymph node transfer in the ESCC group(P>0.05).3. The expression of VEGF-C showed no significant association with age, gender,the degree of differentiation,the deepen of tumor invasion in the ESCC group(P>0.05),but it shows significant association with lymph node metastasis(P<0.05).4. The expression between Annexin-1and VEGF-C shows a negative correlation in the ESCC group.